ErbB Interface Peptidomimetics And Methods Of Use Thereof

ABSTRACT

Peptides, mimetics and antibodies of erbB, TNF, and IgSF receptors and pharmaceutical compositions comprising the same are described. Methods of using such antibodies, peptides, and mimetics in therapeutic, prophylactic, imaging and diagnostic applications are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.10/119,288, filed Apr. 4, 2002, which claims the benefit of ProvisionalApplication No. 60/282,037, filed Apr. 6, 2001, and ProvisionalApplication No. 60/309,864, filed Aug. 3, 2001, each of which isincorporated by reference herein in its entirety.

FIELD OF INVENTION

The present invention is directed to treatments and diagnoses formammalian tumors. More particularly this invention is directed tomethods of preventing, treating, and diagnosing mammalian cancer tumorsusing mimetics and antibodies.

BACKGROUND

Members of the c-erbB (erbB) family of receptor tyrosine kinase genes,including epidermal growth factor receptor c-erbB1 (EGFr, HER1), c-erbB2(HER2, neu, p185), c-erbB-3 (HER3), and c-erbB-4 (HER4), are known to beoncogenes that encode cell surface receptor proteins. The receptors,under some circumstances, display abnormal kinase activities thatcontribute to cell proliferation and transformation.

ErbB family receptor tyrosine kinases (RTKs) form homodimeric,heterodimeric, or perhaps oligomeric complexes that are catalyticallyactive and, thereby, couple extracellular signals with alterations ofcellular growth and differentiation status. Their ligands and subsequentreceptor-mediated signaling have been implicated in survival,proliferation and differentiation in a variety of cell types (reviewedin Dougall et al. 1994; O'Rourke, et al. 1997; Pinkas-Kramarski, et al.1997; Tzahar and Yarden 1998).

All members of the erbB family share structural features, including anextracellular ligand-binding domain that contains four subdomains,including two cysteine-rich subdomains, a single amphipathictransmembrane domain, and an intracellular kinase domain. The kinasedomain shows the highest degree of amino acid sequence similarity (about80%) among members of this family.

Overexpression of erbB receptors has been found in many types of humancancer, raising the possibility that receptor-linked therapies may beuseful as cancer management strategies. EGFr (erbB1) is the mostextensively studied member in this family. The EGFr gene is amplifiedand rearranged in many human brain tumors of glial origin and in somecell lines. Ullrich et al, has found the gene for the EGFr cellularanalogue of the avian vital oncogene v-erb-B. (Ullrich et al, Nature,Vol. 309, pp. 418-425, 1984). The EGFr receptor is a transmembraneglycoprotein of about 170 kDa (Cohen, J. Biol. Chem., Vol. 258, pp.1523-1531, 1982). Overexpression of the EGFr has been found in a varietyof tumors, including bladder, esophagus, lung, glioblastoma and breast.In breast cancers, over 40% of the tumors are EGFr positive, and EGFrlevels negatively correlate with steroid receptor (estrogen receptor andprogesterone receptor) levels. The EGF-receptor exists in two kineticforms (low affinity and high-affinity receptors) that may beinter-convertible. (Fernandez-Pol, Biol. Chem., Vol. 260, pp 5003-5011,1985.) Expression of EGF-receptors has been implicated in theprogression of tumor growth. In addition, an association has beendetected between late stages of melanoma development and extra copies ofthe chromosome carrying the EGFr gene. (Koprowski et al., Somatic Celland Molecular Genetics, Vol. 11, pp. 297-302, 1985.)

A variety of strategies have also been developed for targeting the erbB1receptor including monoclonal antibodies, ligand-linked immunotoxins,tyrosine kinase inhibitors, and antisense approaches. (Zhang et al.,2000.) Of all the members of the erbB family, erbB2 is the mostcorrelated to breast cancer, ovarian cancer and pancreatic cancer.Initially identified in rat neuroglioblastomas induced by a carcinogenethylnitrosourea, neu (also known as her2/erbB2) is a proto-oncogeneencoding a 185 kDa receptor-tyrosine-kinase which is highly homologouswith, but distinct from, EGFr. The translation product of the erbB-2oncogene is p185, a transmembrane glycoprotein having tyrosine kinaseactivity and a molecular weight of about 185,000 daltons as determinedby carrying out electrophoresis on the glycoprotein and comparing itsmovement with marker proteins of known molecular weight. Experimentshave shown that p185 forms homodimers with other p185 molecules orheterodimers with epidermal growth factor receptor EGFR (erbB1) and thatthese dimers exhibit elevated tyrosine kinase activity, which bringsabout the transformed phenotype in cells having such dimers.

Amplification of the erbB2 gene, the human homologue of neu, andsubsequent overexpression of the polypeptide product p185 has beenidentified in 25-30% of primary breast and ovarian cancers, although nooncogenic point mutation has been detected in erbB2 associated withhuman carcinomas. In murine fibroblasts NIH3T3 and NR6, overexpressionof erbB2 results in transformation, indicating that oncogenic mutationis not necessary for erbB2. Previous work has shown that overexpressionof erbB2/neu can lead to oligomers which have enhanced kinase activity.

Overexpression of the erbB2 gene in human breast cancer is alsoassociated with a poor prognosis and resistance to hormonal treatmentand chemotherapy. Advanced stages of malignancy, characterized by largetumor size and increased number of positive lymph nodes as well asreduced survival time and decreased time to relapse, was directlycorrelated with an increased level of amplification of the neu gene. Theneu protooncogene is expressed at low levels in normal human tissues.

c-erbB3 is expressed in a variety of normal tissues of ephithelialorigin and is overexpressed in a subset of human mammary tumors. c-erbB4(erbB3) is most predominantly expressed in several breast carcinoma celllines and also in normal skeletal muscle, heart, pituitary, andcerebellum. The erbB3 receptor has only limited kinase activity.Overexpression of the erbB3 or erbB4 alone cannot transform NIH3T3cells, even in the presence of ligand. It is suggested that thecontribution of erbB3 and erbB4 to tumorigenicity depends onheterodimerization with the EGFr or erbB2.

Monoclonal antibodies (mAbs) and fragments from them have been usedclinically for the diagnosis and treatment of many different humandiseases (Dougall et al 1994, Oncogene 2109-23). The anti-tumor efficacyof mAbs not only requires specificity towards tumor antigens which showenhanced expression in neoplastic tissue, but also must demonstrate thedesired biological effect, namely, the inhibition of tumor growth. U.S.Pat. No. 4,522,918, e.g., discloses a cancer treatment using monoclonalantibodies directed at surface antigens of human mammary adenocarcinomacells.

Capone et al., JNCI 72: 673-677, (1984), investigated the relationshipbetween antigen density and immunotherapeutic response elicited bymonoclonal antibodies against solid tumors. These investigators usedmonoclonal antibodies specific against human breast cancer. It was foundthat passively administered monoclonal antibody can be effective inproducing a tumor regression response against solid tumors. Tumoricidalresponse with monoclonal antibody appeared to be exponentially relatedto the density of the antigen on the cells. U.S. Pat. No. 6,252,050describes methods for generating cross-reactive antibodies. Antibodiesagainst p185 and methods of using such antibodies are described in U.S.Pat. Nos. 6,165,464, 5,772,997, 5,770,195, 5,725,856, 5,720,954, and5,677,171, which are incorporated herein by reference. U.S. Pat. No.5,705,157 describes antibodies against EGFR. U.S. Pat. No. 5,470,571discloses a cancer treatment using monoclonal antibodies directed at theEGFr generated from the A431 carcinoma cell line. Each of theaforementioned U.S. patents is hereby incorporated herein by referencein its entirety.

In the case of receptor-dimerization, a construct containing theextracellular domain plus the transmembrane domain of p185 was able toinitiate the p185-EGFr dimerization (Qian et al, PNAS 91, 1500, 1994).Later, an alternative transcript product of p185 with only subdomain Iand II was found to be able to dimerize with p185 (Doherty et al. PNAS1999, 96, 10869).

An approach for disabling receptor activity is to target protein-proteininteractions involved in receptor functioning. Since protein-proteininteractions play a key role in various mechanisms of cellular growthand differentiation, and viral replication, inhibition of theseinteractions is a promising novel approach for rational drug designagainst a wide number of cellular and viral targets (Zutshi et al., CurrOpin Chem Biol 1998, 2, 62-66; Peczuh et al., Chem. Rev. 2000, 100,2479-2494). Binding of polypeptide hormones, growth factors or cytokinesto cell-surface receptors activates dimerization (oligomerization) ofthe receptors which leads to the signal transduction to the interior ofthe cell (Heldin, Cell 1995, 80, 213-223). While most of the receptorinhibitors developed to date have been focused on the blockade ofreceptor-ligand or enzyme-substrate interactions, repression ofreceptor-receptor interactions that accompany oligomerization alsorepresents an important objective for disabling receptor functioning.

Although ligand-induced homo- and heterodimerization of the full-lengthnative erbB receptors has been established and well documented,experimental data on self-associations of the extracellular domains ofthese receptors is somewhat contradictory. In analytical ultracentrifugation and MALLS studies, ligand-induced homodimerization hasbeen demonstrated for erbB1 and erbB4 (Ferguson et al., EMBO J. 2000,19, 4632-4643).

However, no homo-oligomerization could be observed for the erbB3receptor and the only erbB receptor combinations that producedheterodimers in the presence of HRGβ1 were erbB2/erbB4 and to a smallerextent erbB2/erbB3. In contrast, both erbB3 homodimerization anderbB3/erbB2 heterodimerization have been reported for the ectodomains,but these effects could only be observed when ectodomains of thereceptors were anchored to the membrane (Tzahar et al., EMBO J. 1997,16, 4938-4950). Landgraf and Eisenberg have reported ligand-independentself-association of erbB3 ectodomains that could be disrupted by HRGβ1(Landgraf et al., Biochemistry 2000, 39, 8503-8511). Both monomeric andoligomeric forms of erbB3 were detected in the presence of HRGβ1 bysize-exclusion chromatography. Addition of the ligand produced a shifttoward a low-molecular mass species.

The present inventors have identified distinct extracellular subdomainsof erbB2 that are involved in heterodimerization with erbB1 (Kumagai etal, Proc Natl Acad Sci USA 2001, 98, 5526-5531). Peptidomimetics againstsubdomain IV alter the heteromeric signaling and transforming activitiesinduced by EGF after associating with EGFR. Peptidometics and antibodiesthat target subdomain W are therefore useful as therapeutic agentsagainst erbB-expressing tumors.

SUMMARY

Certain embodiments of the present invention relate to antibodies whichbind to assembly epitopes of erbB1, erbB2, erbB3, erbB4 and assemblyepitopes of TNF receptors.

Certain embodiments of the present invention relate to antibodies whichblock the oligomerization of receptors. In some preferred embodiments,the antibodies are induced by immunizing with a peptide or proteinsubdomain containing structural elements involved in theoligomerization. In some embodiments, the structural element is acystine knot.

Certain embodiments of the present invention relate to antibodies whichbind to subdomains of erbB1, erbB2, erbB3, erbB4, TNF receptors, ormembers of the IgSF, or assemblies thereof, that contain cystine knots.

Certain embodiments of the present invention relate to antibodies whichbind to cystine knots of erbB1, erbB2, erbB3, and erbB4, or to cystineknots of TNF receptors.

Certain embodiments of the present invention relate to pharmaceuticalcompositions comprising which binds to erbB receptors or to TNFreceptors. Some embodiments relate to injectable pharmaceuticals whichare sterile and pyrogen free.

Certain embodiments of the present invention relate to antibodies whichbind to interaction surfaces in the extracellular domains of erbBreceptors or to interaction surfaces in the extracellular domains of TNFreceptors.

Certain embodiments of the present invention relate to peptides whichmimic erbB receptors or TNF receptors. In some preferred embodiments,the peptides mimic an extracellular domain of an erbB receptor or of anextracellular domain of a TNF receptor. In more preferred embodiments,the peptides mimic subdomain W of the erbB receptor. In even morepreferred embodiments, the peptides mimic the S22 or S23 loop of theerbB receptor.

Certain embodiments of the present invention relate to mimetics of erbBreceptors or TNF receptors. In some preferred embodiments, the mimeticis a mimetic of an extracellular domain of an erbB receptor or of anextracellular domain of a TNF receptor. In more preferred embodiments,the mimetic is a mimetic of subdomain W of the erbB receptor. In evenmore preferred embodiments, the mimetic is a mimetic of the S22 or S23loop of the erbB receptor.

Certain embodiments of the present invention relate to pharmaceuticalcompositions comprising peptides or mimetics of erbB receptors or TNFreceptors, in combination with anti-cancer drugs. Some such embodimentsare injectable pharmaceuticals which are sterile and pyrogen free.

Certain embodiments of the present invention relate to methods oftreating human patients having solid tumors by administering to thepatient peptides or mimetics of or antibodies to erbB receptors. In somesuch embodiments the administration of peptides, mimetics or antibodiesmay optionally be followed by exposing the patient to a therapeuticallyeffective amount of anti-cancer radiation. In some other embodiments,the administration of peptides, mimetics, or antibodies is performed incombination with administration of a therapeutically effective amount ofa chemotherapeutic agent. In some embodiments the administration ofpeptides, mimetics or antibodies is performed in combination withadministration of a therapeutically effective amount of achemotherapeutic agent and followed by exposing the patient to atherapeutically effective amount of anti-cancer radiation.

Certain embodiments of the present invention relate to methods oftreating human patients having solid tumors by administering to thepatient peptides or mimetics of erbB receptors that are conjugated toradioactive, chemotherapeutic, or photodynamic therapeutic agents.

Certain embodiments of the present invention relate to pharmaceuticalcompositions comprising antibodies which bind to erbB or TNF receptorsthat are conjugated to radioactive or chemotherapeutic agents. Someembodiments relate to injectable pharmaceuticals which are sterile andpyrogen free.

Certain embodiments of the present invention relate to methods ofpreventing tumors in human patients by administering to the patientanti-erbB antibodies.

Certain embodiments of the present invention relate to methods ofpreventing tumors in human patients by administering to the patientpeptide or mimetics of erbB receptors.

Certain embodiments of the present invention relate to methods ofimaging erbB tumors in human patients having such tumors usingdetectable anti-erbB antibodies.

Certain embodiments of the present invention relate to pharmaceuticalcompositions comprising detectable antibodies which bind to erbBreceptors, to TNF receptor, or to members of the IgSF. Some embodimentsrelate to injectable pharmaceuticals which are sterile and pyrogen free.

Certain embodiments of the present invention relate to diagnostic kitsand to methods for imaging and/or detecting solid tumors using anti-erbBantibodies.

Certain embodiments of the present invention relate to a method ofpreventing tumors in a mammal which comprises administering to saidmammal an agent selected from the group consisting of a peptideconsisting essentially of an erbB subdomain IV, a peptide consistingessentially of an erbB subdomain IV peptide wherein between 1-10 aminoamino acids of the subdomain have been substituted with a conservativeamino acid, and a peptide consisting essentially of between 10-25contiguous amino acids of a an erbB subdomain IV.

Certain embodiments of the present invention relate to a vaccine forpreventing tumors in a mammal which comprises an active agent selectedfrom the group consisting of a peptide consisting essentially of an erbBsubdomain IV, a peptide consisting essentially of an erbB subdomain Wpeptide wherein between 1-10 amino amino acids of the subdomain havebeen substituted with a conservative amino acid, and a peptideconsisting essentially of between 10-25 contiguous amino acids of a anerbB subdomain IV and an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B represents the schematic representation of the expressionvectors and heterodimer formation between EGFr and Neu mutants. FIG. 1Adepicts the schematic representation of the expression vectors. FIG. 1Bdepicts heterodimer formation between EGFr and Neu mutants.

FIG. 2A-C. Molecular model of ErbB-2: Second cysteine rich domainproximal to transmembrane (S22) is shown (A). This module adopts asimilar topology as that of the TNF receptor. Peptides designed fromA2-like domains are exocyclic (S22-AFA; B) while peptides from B2-likedomains (S23-BPT; C) are fashioned as full cystine knots.

FIG. 3A-B. Surface plasmon resonance analysis of the interaction betweenthe B2-S22-APE peptide and the ectodomain of erbB2 (A) or erbB2-SbdIV(B). Dose response.

FIG. 4. Surface plasmon resonance analysis of the inhibitory effect ofthe B2-S22-AFA peptide on the heregulin-induced oligomerization of erbBreceptors. ErbB1, erbB2, and erbB3 were immobilized on the surface chipand 300 nM erbB3 was injected either alone (1A, 2A, 3A) or in thepresence of 5 μM HRGβ1 (1C, 2B, 3C). Traces 1B, 2B and 3B show bindingof erbB3 to the immobilized receptors in the presence of HRGβ1 afterpre-injection of B2-S22-AFA at 10 μM.

FIG. 5. Inhibitory effect of the B2-S22-AFA and B3-S22-APQ peptides onthe heregulin activation and dimerization of ErbB receptor. (A)32D-E2/E4 cells were incubated in the absence (−) or presence (+) of 10μg/ml HRGβ1 and 10 μg/ml erbB peptides and analyzed by chemicalcross-linking and immunoblotting experiments as described in theExperimental Procedures. (B) Dose dependence of the inhibitory effect bythe B2-S22-AFA peptide.

FIG. 6. Inhibitory activity of the erbB peptides against the growth ofT6-17 cells studied in an MTT assay.

FIG. 7A-B. Correlation between receptor-binding properties of the erbBpeptides and their biological activity against erbB2-overexpressingT6-17 cells. Plots show correlation between peptides' activities in MTTassays and (A) their receptor binding affinities, or (B) theirinhibitory activity against erbB receptor oligomerization.

FIG. 8A-B. Inhibitory effect of the ErbB peptides on cell survival ofthe 32D cell transfectants. 32D cell transfectants were grown in eitherthe ErbB ligand medium (A) or the 11-3 supplement (WEHI) medium (B), andcell viability was determined as described in the ExperimentalProcedures. In each experiment, the standard error did not exceed 10%.

FIG. 9A-B. Molecular modeling of erbB1 homo-dimerization. Possiblearrangement of the erbB1-EGF (2:2) complex according to model 1 (EGFcross-links two erbB1 monomers, A) and model 2 (EGF binds to one erbB1monomer, B).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In some embodiments, of the present invention compositions comprisingpeptides and mimetics of and antibodies to regions of receptors whichfacilitate oligomerization are provided, preferably directed to regionsof receptors comprising one or more cystine knots. The present inventionfurther provides methods for treatment, diagnosis, and imaging ofmammalian tumors using such peptides and antibodies.

DEFINITIONS

as Used Herein, the Term “Erbb” Refers to Receptors in the Erbb Familyof Receptor tyrosine kinases which assemble into hetero- or homodimers,including, but not limited to, erbB1 (EGFr—epidermal growth factorreceptor, HER1), erbB2 (neu, p185, HER2), erbB3 (HER3), and erbB4(HER4).

As used herein, the term “TNF” refers to receptors that bind tumornecrosis factor-like ligomers which assemble into oligomers. TNFreceptors include, but are not limited to, TNF, FAS, RANK, TRAIL, andCD40.

As used herein, the terms “p185/EGFr cancer”, “p185/EGFr tumors”,“erbB2/EGFr cancer” and “erbB2/EGFr tumors” are meant to refer to tumorcells and neoplasms which express erbB2 and EGFr. erbB2/EGFr tumors havep185 and EGF receptors on their cell surfaces.

As used herein, the terms “erbB tumor”, and “erbB cancer” are meant torefer to tumor cells and neoplasms which express one or more erbBreceptors. Some erbB tumor cells or neoplasms may express p185 receptorson their cell surfaces.

As used herein, the terms “TNF”, and “TNF-related pathologies” are meantto refer to pathologies that involve one or more TNF family receptors.

As used herein, the term “oligomerization” refers to the process bywhich assemblies of monomers are formed into multimers. Examples ofassemblies formed through this process include but are not limited todimers, trimers and tetramers, etc. Such assemblies may comprise two ormore identical monomers yielding a homodimer, homotrimer, homotetramer,etc., or two or more different monomers yielding a heterodimer,heterodimer, heterotrimer, etc.

As used herein, the term “antibody” is meant to refer to antibodies, aswell as antibody fragments such as FAb and F(Ab)₂ fragments, recombinantantibodies or recombinant antibody fragments. Antibodies may, in somepreferred embodiments, be monoclonal, humanized, primatized, or chimericantibodies.

As used herein, the term “cystine knot” refers to a polypeptide formedby at least two disulfide bonds and the protein chains linking them,penetrated by a third disulfide bond and is further described in Murzinet al., J. Mol. Biol. 247: 536-540, which is incorporated by referencein its entirety. For example, in the TNF receptor family a cystine knotconsists of 42 amino acid residues with 6 cysteine residues forming 3inter-chain disulfide bonds to create the structural motif.

As used herein, the phrase “cystine knot specific antibody” refers to anantibody which binds to a cysteine-rich domain, a cystine knot, or aportion of a cystine knot loop. In some embodiments, the antibody bindsto cysteine bonded loops found within a cysteine rich domain.

As used herein, the phrase “cystine knot comprising region” refers to aportion of a receptor that comprises one or more cystine knots. In someembodiments, the cystine knot-comprising region is an extracellularportion of the receptor. In some preferred embodiments, the cystine knotcomprising region is subdomain IV. In more preferred embodiments, thecystine knot comprising region is selected from the group consisting ofthe S22 loop and the S23 loop.

As used herein, the term “region” refers to a part of a receptorcomprising at least one portion. Representative receptor regionsinclude, but are not limited to, extracellular regions, transmembraneregions, and intracellular regions.

As used herein, the term “portion” refers to at least 3-5 amino acids,more preferably at least 8-10 amino acids, more preferably at least11-15 amino acids, more preferably at least 17-24 amino acids, and evenmore preferably at least 25-30 amino acids, and most preferably at least30-45 amino acids.

As used herein, the term “conformation site” refers to a site on areceptor which affects the conformation of the receptor. In someembodiments, binding of an antibody, peptide or mimetic to theconformation site changes the conformation of the receptor such thatoligomerization of the receptor is prevented and, preferably, reduces oreliminates receptor signaling. In some preferred embodiments, theconformation site is a receptor-receptor contact point.

As used herein, the term “mimetic” is used to refer to compounds whichmimic the activity of peptide. Mimetics are non-peptides but maycomprise amino acids linked by non-peptide bonds. U.S. Pat. No.5,637,677 and its parent applications contain detailed guidance on theproduction of mimetics. Briefly, the three dimensional structure of thepeptides which specifically interacts with the three dimensionalstructure of erbB receptors is duplicated by a molecule that is not apeptide.

As used herein, the terms “conformationally restricted peptides”,“constrained peptides” and “conformationally constrained peptides” areused interchangeably and are meant to refer to peptides which, forexample through intramolecular bonds, are conformationally stable andremain in a sufficiently restricted conformation. The compounds have anaffinity to erbB receptors and, when bound to erbB receptors on cells,have a biologically active effect on cells that have a erbB-mediatedtransformation phenotype.

As used herein, the terms “aromatic amino acids” and “aromatic aminoacid residues” used interchangeably are meant to refer to phenylalanineand tyrosine.

As used herein, the term “exocyclic amino acid residue” is meant torefer to amino acid residues which are linked to cyclicized peptide butwhich are not within the portion of the peptide that makes up thecircularized structure.

As used herein, the term “exocyclic portions” is meant to refer to anamino acid sequence having one or more amino acid residues which arelinked to cyclicized peptide but which are not within the portion of thepeptide that makes up the circularized structure.

As used herein, the term “linking moiety” is meant to refer to amolecular component or functional group which is capable of formingbonds with three amino acids.

As used herein, the term “linking amino acid residue” is meant to referto an amino acid residue that is a linking moiety.

As used herein, the terms “active sequence” and “active region” are usedinterchangeably and are meant to refer to the amino acid sequence of theportion of a compound of the invention that is directly interacts withan erbB receptor, wherein the interaction is characterized by anaffinity between the active portion and an erbB receptor.

In some embodiments, the peptides and mimetics are constrained mimics ofthe loops or repeats present in subdomain IV of erbB receptors. In someembodiments, the peptides and mimetics mimic a cystine knot comprisingregion, preferably a cystine knot or portion thereof. In someembodiments, binding of a peptide or mimetic to an erbB or TNF receptorprevents dimerization of the receptor, and, preferably, reduces oreliminates receptor signaling.

As used herein, the term “dimerization site” is used interchangeablywith the terms “interaction site” and “interaction surface” and refersto a site on a receptor that forms a bond with another receptor when thetwo receptors dimerize. In some embodiments, the dimerization site isligand-independent, i.e., the dimerization site is not dependent on thepresence or absence of a particular bound ligand. In other embodiments,the dimerization site is ligand-dependent, i.e., the dimerization siteis dependent on the presence or absence of a particular bound ligand. Insome embodiments, binding of an antibody, peptide or mimetic to thedimerization site prevents dimerization of the receptors and,preferably, reduces or eliminates receptor signaling. In some preferredembodiments, the dimerization site is subdomain W of the erbB receptoror a portion thereof.

As used herein, the term “assembly” is used interchangeably with“ensemble” or “dimer” and refers to homo- or heterooligomers ofreceptors. Such assemblies may comprise erbB1, erbB2, erbB3, erbB4, orcombinations thereof, or receptors in the TNF family of receptors,including but not limited to FAS, RANK, TRAIL, and CD40, or combinationsthereof, or members of the IgSF superfamily, including but not limitedto ICOS and CD28.

As used herein, the term “high risk individual” is meant to refer to anindividual who has had an erbB or TNF related pathology or pathologiesassociated with IgSF members either removed or in remission, and who istherefore susceptible to a relapse or recurrence. In the case of erbB,as part of a treatment regimen for a high risk individual, theindividual can be prophylactically treated against tumors that they havebeen diagnosed as having had in order to combat a recurrence. Thus, onceit is known that an individual has had an erbB-cancer, the individualcan be treated according to the present invention to prevent normalcells from transforming into tumor cells.

As used herein, the term “in combination with” is meant to refer toadministration of the antibody, peptide or mimetic compositions of theinvention with radiation therapy and/or chemotherapy. Administration ofthe antibody, peptide or mimetic compositions may take place prior to,simultaneously with, or after radiation therapy and/or chemotherapy.

As used herein, the term “therapeutically effective amount” is meant torefer to an amount of an antibody, peptide or mimetic which produces amedicinal effect observed as reduction or reverse in tumorigenicphenotype of tumor cells in an individual when a therapeuticallyeffective amount of a antibody is administered to the individual.Therapeutically effective amounts are typically determined by the effectthey have compared to the effect observed when a composition whichincludes no active ingredient is administered to a similarly situatedindividual.

As used herein, the term “preventing the development of tumors” is meantto refer to preventing the transformation of normal cells into tumorcells including inhibiting the transformation of cells that have anormal or incomplete transformed phenotype into fully transformedphenotype. Thus, the development of tumors refers to the transformationevent which results in the acquisition of a transformed phenotype.According to some aspects of the present invention, antibodies, peptidesor mimetics may be administered to individuals who are at risk ofdeveloping tumors. The prophylactic administration of an antibody,peptide or mimetics to high-risk individuals results in the preventionof the transformation event occurring. Cells having the normal phenotypeare not converted to the cells having transformed phenotype. Theantibodies, peptides, or mimetics prevent tumors before they are formedby preventing a normal cell from becoming a cancer cell.

As used herein, the term “prophylactically effective amount” is meant torefer to an amount of an antibody, peptide, or mimetic which produces amedicinal effect observed as the prevention of non-transformed cellsfrom becoming transformed in an individual when a prophylacticallyeffective amount of an antibody, peptide or mimetic is administered toan individual. Prophylactically effective amounts are typicallydetermined by the effect they have compared to the effect observed whena composition which includes no active ingredient is administered to asimilarly situated individual.

As used herein, the phrase “injectable pharmaceutical composition”, orvariants thereof, refers to pharmaceutical compositions which satisfythe USP requirements for “injectables”, i.e., sterile, pyrogen- andparticulate free, and possessing specific pH and isotonicity values.

Antibodies

In addition to molecules designed from assembly epitopes of erbB1,erbB2, erbB3 erbB4, and assembly epitopes of TNF or IgSF receptors, thepresent invention comprises molecules, including but not limited toantibodies and peptide mimetics, based on interacting surfaces ofreceptors.

In some embodiments, antibodies are specific to dimerization sites oferbB, TNF, or IgSF receptors. In some embodiments, antibodies arespecific to a dimerization site of erbB1 or erbB2. Binding of theantibody to the dimerization site may prevent the dimerization of thereceptor, and, preferably, reduces or eliminates receptor signaling.

In some embodiments, antibodies are specific to conformation sites oferbB, TNF, or IgSF receptors. In a preferred embodiment, antibodies arespecific to a conformation site of erbB1 or erbB2. In some embodiments,antibodies are specific to a conformation site of erbB1 or erbB2.Binding of the antibody to the conformation site changes theconformation of the receptor such that the receptor is not able todimerize, and, preferably, reduces or eliminates receptor signaling.

In some embodiments, antibodies are provided which bind to cystine knotcomprising regions or cystine knots or portions thereof of erbB, TNF, orIgSF receptors. In some embodiments, antibodies are specific for cystineknot in an extracellular domain of an erbB, TNF, or IgSF receptor.

Peptides, Mimetics and Antibodies

The present invention describes inter alia the biochemical consequencesof peptides mimetics which are capable of disabling the assembly of erbBreceptor polypeptides or TNF receptor polypeptides by differentmechanisms.

The present invention relates, inter alia, to constrained peptides thatcontain exocyclic portions including exocyclic amino acids that arearomatic amino acids as well as an active region which specificallybinds to erbB. Examples of constrained peptides are found in U.S. Pat.No. 6,100,377.

The present invention also relates to mimetics which specifically bindto erbB.

The present invention is useful to therapeutically treat an individualidentified as suffering from erbB tumors in order to reverse thetransformed phenotype of the tumor cells. The present invention isuseful to prophylactically treat an individual who is predisposed todevelop an erbB tumors or who has had erbB-associated tumors and istherefore susceptible to a relapse or recurrence. The present inventionis useful to detectably image tumors with respect to erbB receptors ontheir surfaces. The present invention is useful to detect and quantifyerbB on all surfaces.

According to some embodiments, the present invention provides peptideshaving the formula:

R₁-R₂-R₃-R₄-R₅-R₆-R₇

wherein:

-   -   R₁ is 1-6 amino acid residues, at least one of which is tyrosine        or phenylalanine;    -   R₂ is a linking moiety which bonds with R₁, R₃ and R₆ such that        a portion of said peptide is cyclicized;    -   R₃ is 0-20 amino acids;    -   R₄ is 6-12 amino acids;    -   R₅ is 0-20 amino acids;    -   R₆ is a linking moiety which bonds with R₅, R₇ and R₂ such that        a portion of said peptide is cyclicized;    -   R₇ is 1-6 amino acid residues, at least one of which is tyrosine        or phenylalanine;    -   wherein: R₁, R₂, R₃, R₄, R₅, R₆ and R₇, taken together, are 30        amino acids or less.

In some embodiments, R₄ comprises F-P-D-E-E-G-A (SEQ ID NO:1). In someembodiments, R₄ consists of F-P-D-E-E-G-A (SEQ ID NO:1). In someembodiments, R₄ comprises F-Y-P-D-E-E-G-A (SEQ ID NO:2). In someembodiments, R₄ consists of F-Y-P-D-E-E-G-A (SEQ ID NO:2).

The primary function of R₁ in compounds of the present invention arisesfrom the presence of at least one amino acid that contains an aromaticgroup: i.e. the presence of tyrosine or phenylalanine. The presence ofthe aromatic amino acid at position R₁ results in an increase affinityof the peptide to erbB and an attendant increase in activity of thecompound. In embodiments where additional amino acid residues arepresent, they can present the aromatic amino acid in a more effectiveposition to further increase the affinity and activity of the compound.Additional amino acids that may be present must not eliminate the effectthat the aromatic amino acid has on affinity or activity. Examples ofamino acid sequences which may be used as R₁ are disclosed in co-pendingU.S. Pat. No. 6,100,377. In some embodiments, the additional amino acidsare present as a site for linkage to detectable labels or moieties. Insome embodiments, the additional amino acids are present as a site fordimerization with other peptides; either for formation of homodimerswith each other or heterodimers with other peptides.

In some preferred embodiments, R₁ is 1-5 amino acids. In some preferredembodiments, R₁ is 4 amino acids. In some preferred embodiments, R₁ is 3amino acids. In some preferred embodiments, R₁ is 2 amino acids. In somepreferred embodiments, R₁ is 1 amino acid. In some preferredembodiments, R₁ comprises S-Y. In some preferred embodiments, R₁consists of S-Y. In some preferred embodiments, R₁ comprises G-S-Y. Insome preferred embodiments, R₁ consists of G-S-Y. In some preferredembodiments, R₁ consists of Y. In some preferred embodiments, R₁consists of K. In some preferred embodiments, R₁ comprises K. Otherexamples of R₁ include G-G-S-Y (SEQ ID NO:21) and G-G-G-S-Y (SEQ IDNO:22). Contemplated equivalents include aromatic functional groups atR₁ which are not part of tyrosine or phenylalanine.

The function of R₂ is to form bonds with R₁ as well as to form bondswith R₆ which cyclicize or otherwise conformationally restrict themolecule. Bonds between R₂ and R₆ cyclicize the molecule and therebymaintain R₃-R₄-R₅, and, specifically R₄, in a constrained conformationthat produces the specific biologically active surface that has anaffinity for and interacts with erbB. Further, in such an arrangement R₁becomes an exocyclic portion of the peptide. Accordingly, R₂ may be anymoiety capable of forming bonds with R₆ as well as R₁ and R₃.

R₂ is preferably an amino acid residue, preferably cysteine. When bothR₂ and R₆ are cysteine, the disulfide bonds form between the twocysteines cyclicize the molecule. It is contemplated that R₂ may anymoiety that, together with R₆, will allow for the cyclization of theportion of the molecule that includes R₁-R₂-R₃-R₄-R₅-R₆ while renderingR₁ and R₇ exocyclic portions of the peptide. Those having ordinary skillin the art can readily prepare peptides according to the presentinvention in which R₂ and R₆ are moieties capable of forming bonds toeach other. The cyclization of linear peptides using disulfide bondsbetween non-adjacent cysteines is well known. Similarly, othernon-adjacent amino acid residues may be linked to cyclicize a peptidesequence and the means to do so are similarly well known. Other methodsof cyclization include those described by Di Blasio, et al., (1993)Biopolymers, 33:1037-1049; Wood, et al., (1992) J. Pep. Prot. Res.,39:533-539; Saragovi, et al., (1992) Immunomethods, 1:5-9; Saragovi, etal., (1991) Science, 253:792-795; Manning, et al., (1993) Reg. Peptides,45:279-283; Hruby, (1993) Biopolymers, 33:1073-1082; Bach, et al.,(1994) New Adv. Peptidomimetics Small Mol. Design, I:1-26; andMatsuyama, et al., (1992) J. Bacteriol., 174:1769-1776, each of whichare incorporated herein by reference.

The function of R₃ is to serve as a spacer and provide structure topresent the active region in proper conformation. In some embodiments,the cyclization of the active region by particular linking moietiesresults in the proper folding of the active region to place it in activeconformation and no R₃ is required. In some embodiments, the cyclizationof the active region by particular linking moieties requires additionalspacing and turns to facilitate that proper folding of the active regionin order to place it in active conformation. In such embodiments, aminoacid residues or sequences may be provided at R₃. In some preferredembodiments, R₃ is 0-10 amino acids. In some preferred embodiments, R₃is 0-5 amino acids. In some preferred embodiments, R₃ is 0 amino acids.

R₄ is the active region of the compounds according to this aspect of theinvention. In compounds of the invention, the functional groups of theactive region are in a conformation which places them in a particularthree dimensional arrangement that allows them to interact with theamino acids and functional groups thereon of an erbB receptor and tobind to an erbB receptor through such interactions. In peptide mimetics,the functional groups are provided in the active three-dimensionalarrangement but are connected to modified or different backbones. It ispossible to vary each residue with one that contributes equivalent bulkand hydrophobic moment and that still permits hydrogen bonding tosurrounding water molecules or to residues to which the compoundattaches.

The function of R₅ is to present the active region in properconformation. In some embodiments, the cyclization of the active regionby particular linking moieties results in the proper folding of theactive region to place it in active conformation and no R₅ is required.In some embodiments, the cyclization of the active region by particularlinking moieties requires additional spacing and turns to facilitatethat proper folding of the active region in order to place it in activeconformation. In such embodiments, amino acid residues or sequences maybe provided at R₅. In some preferred embodiments, R₅ is 0-10 aminoacids. In some preferred embodiments, R₅ is 0-5 amino acids. In somepreferred embodiments, R₅ is 0 amino acids.

The function of R₆ is to form bonds with R₂ which cyclicize or otherwiseconformationally restrict the molecule. Bonds between R₆ and R₂cyclicize the molecule and thereby maintain R₃-R₄-R₅, and, specificallyR₄, in a constrained conformation that produces the specificbiologically active surface that has an affinity for and interacts witherbB. Accordingly, R₆ may be any moiety capable of forming bonds with R₂as well as R₅ and R₇. R₆ is preferably an amino acid residue, preferablycysteine. When both R₆ and R₂ are cysteine, disulfide bonds formedbetween the two cysteines cyclicize the molecule. It is contemplatedthat R₆ may any moiety that, together with R₂, will allow for thecyclization of the molecule. Those having ordinary skill in the art canreadily prepare peptides according to the present invention in which R₂and R₆ are moieties capable of forming bonds to each other. Thecyclization of linear peptides using disulfide bonds betweennon-adjacent cysteines is well known. Similarly, other non-adjacentamino acid residues may be linked to cyclicize a peptide sequence andthe means to do so are similarly well known. Other methods ofcyclization include those described by Di Blasio, et al., (1993)Biopolymers, 33:1037-1049; Wood, et al., (1992). J. Pep. Prot. Res.,39:533-539; Saragovi, et al., (1992) Immunomethods, 1:5-9; Saragovi, etal., (1991) Science, 253:792-795; Manning, et al., (1993) Reg. Peptides,45:279-283; Hruby, (1993) Biopolymers, 33:1073-1082; Bach, et al.,(1994) New Adv. Peptidomimetics Small Mol. Design, I:1-26; andMatsuyama, et al., (1992) J. Bacteriol., 174:1769-1776, each of whichare incorporated herein by reference.

The primary function of R₇ in compounds of the present invention arisesfrom the presence of at least one amino acid that contains an aromaticgroup: i.e. the presence of tyrosine or phenylalanine. The presence ofthe aromatic amino acid at position R₇ results in an increase in theaffinity of the peptide to erbB and an attendant increase in activity ofthe compound. In embodiments where additional amino acid residues arepresent, they can present the aromatic amino acid in a more effectiveposition to further increase the affinity and activity of the compound.Additional amino acids that may be present must not eliminate the effectthat the aromatic amino acid has on affinity or activity. Examples ofamino acid sequences which may be used as R₇ are disclosed in U.S. Pat.No. 6,100,377.

In some embodiments, the additional amino acids are present as a sitefor linkage to detectable labels or moieties. In some embodiments, theadditional amino acids are present as a site for dimerization with otherpeptides; either for formation of homodimers with each other orheterodimers with other peptides.

In some preferred embodiments, R₇ is 1-5 amino acids. In some preferredembodiments, R₇ is 4 amino acids. In some preferred embodiments, R₇ is 3amino acids. In some preferred embodiments, R₇ is 2 amino acids. In somepreferred embodiments, R₇ is 1 amino acid. In some preferredembodiments, R₇ comprises Y-G-G-S (SEQ ID NO:27). In some preferredembodiments, R₇ consists of Y-G-G-S (SEQ ID NO:27). In some preferredembodiments, R₇ comprises Y-G-G-G (SEQ ID NO:23). In some preferredembodiments, R₇ consists of Y-G-G-G (SEQ ID NO:23). In some preferredembodiments, R₇ comprises Y-G-G-G-S (SEQ ID NO:24). In some preferredembodiments, R₇ consists of Y-G-G-G-S (SEQ ID NO:24). In some preferredembodiments, R₇ comprises Y. In some preferred embodiments, R₇ consistsof Y. In some preferred embodiments, R₇ comprises Y-G-G. In somepreferred embodiments, R₇ consists of Y-G-G. Another example of R₇ isY-G. Contemplated equivalents include aromatic functional groups at R₇which are not part of tyrosine or phenylalanine.

In some preferred embodiments, R₁ and R₇ collectively contain bothtyrosine and phenylalanine. That is, if R₁ comprises tyrosine then R₇comprises phenylalanine and if R₁ comprises phenylalanine then R₇comprises tyrosine. In some preferred embodiments, R₁ and R₇ do not bothcontain tyrosine or phenylalanine. That is, if R₁ comprises tyrosine andnot phenylalanine then R₇ comprises phenylalanine and not tyrosine andif R₁ comprises phenylalanine and not tyrosine then R₇ comprisestyrosine and not phenylalanine.

In some preferred embodiments, R₁, R₂, R₃, R₄, R₅, R₆ and R₇, takentogether, are less than 30 amino acids. In some preferred embodiments,R₁, R₂, R₃, R₄, R₅, R₆ and R₇, taken together, are 20 amino acids orless. In some preferred embodiments, R₁, R₂, R₃, R₄, R₅, R₆ and R₇,taken together, are less than 20 amino acids. In some preferredembodiments, R₁, R₂, R₃, R₄, R₅, R₆ and R₇, taken together, are 15 aminoacids. In some preferred embodiments, R₁, R₂, R₃, R₄, R₅, R₅ and R₇,taken together, are less than 15 amino acids. In some preferredembodiments, R₁, R₂, R₃, R₄, R₅, R₆ and R₇, taken together, are 14 aminoacids. In some preferred embodiments, R₁, R₂, R₃, R₄, R₅, R₆ and R₇,taken together, are 13 amino acids. In some preferred embodiments, R₁,R₂, R₃, R₄, R₅, R₆ and R₇, taken together, are 12 amino acids. In somepreferred embodiments, R₁, R₂, R₃, R₄, R₅, R₆ and R₇, taken together,are 11 amino acids. In some preferred embodiments, R₁, R₂, R₃, R₄, R₅,R₆ and R₇, taken together, are 10 amino acids.

In certain embodiments R₁ is Phe, Tyr, Phe-Glu, or Tyr-Glu; R₂ iscysteine or penicillamine; R₃ is a bond; R₄ is SEQ ID NO:1 or SEQ IDNO:2; R₅ is a bond; R₆ is cysteine or penicillamine; and R₇ is Phe orTyr.

In certain embodiments, R₁ is Phe, Tyr, Phe-Glu, or Tyr-Glu; R₂ iscysteine or penicillamine; R₃ is a bond; R₄ is SEQ ID NO:1; R₅ is abond; R₆ is cysteine or penicillamine; and R₇ is Phe or Tyr.

In certain embodiments, R₁ is Phe, Tyr, Phe-Glu, or Tyr-Glu; R₂ iscysteine or penicillamine; R₃ is a bond; R₄ is SEQ ID NO:2; R₅ is abond; R₆ is cysteine or penicillamine; and R₇ is Phe or Tyr.

In certain embodiments, R₁ is Tyr, Phe-Glu, or Tyr-Glu; R₂ is cysteineor penicillamine; R₃ is a bond; R₄ is SEQ ID NO:2; R₅ is a bond; R₆ iscysteine or penicillamine; and R₇ is Phe or Tyr.

In certain embodiments, the peptide is selected from the groupconsisting of: Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO:3);S-Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO:4); G-S-Y-C-F-P-D-E-E-G-A-C-Y (SEQ IDNO:5); G-G-S-Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO:6);G-G-G-S-Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO:7); Y-C-F-P-D-E-E-G-A-C-Y-G(SEQ ID NO:8); Y-C-F-P-D-E-E-G-A-C-Y-G-G (SEQ ID NO:9);Y-C-F-P-D-E-E-G-A-C-Y-G-G-G (SEQ ID NO:10);Y-C-F-P-D-E-E-G-A-C-Y-G-G-G-S (SEQ ID NO:11); Y-C-F-Y-P-D-E-E-G-A-C-Y(SEQ ID NO:12); S-Y-C-F-Y-P-D-E-E-G-A-C-Y (SEQ ID NO:13);G-S-Y-C-F-Y-P-D-E-E-G-A-C-Y (SEQ ID NO:14);G-G-S-Y-C-F-Y-P-D-E-E-G-A-C-Y (SEQ ID NO:15);G-G-G-S-Y-C-F-Y-P-D-E-E-G-A-C-Y (SEQ ID NO:16);Y-C-F-Y-P-D-E-E-G-A-C-Y-G (SEQ ID NO:17); Y-C-F-Y-P-D-E-E-G-A-C-Y-G-G(SEQ ID NO:18); Y-C-F-Y-P-D-E-E-G-A-C-Y-G-G-G (SEQ ID NO:19); andY-C-F-Y-P-D-E-E-G-A-C-Y-G-G-G-S (SEQ ID NO:20); YCFPDEEGACYK (SEQ ID NO:25); and YCFPDEEGACYGGS (SEQ ID NO: 26). Other peptides are includedwithin the scope of the present invention comprising differentcombinations of R₁, R₂, R₃, R₄, R₅, R₆ and R₇.

In some embodiments, terminal residues of the peptides are modified. Insome embodiments, the terminal residue of R₁ is modified with —OH. Insome embodiments, the terminal residue of R₁ is modified with —NH₂. Insome embodiments, the terminal residue of R₇ is modified with —OH. Insome embodiments, the terminal residue of R₇ is modified with —NH₂.

According to some embodiments,

-   -   R₁-R₂-R₃-R₄-R₅-R₆-R₇        together form a peptide wherein:    -   R₁ is 1-3 amino acid residues, at least one of        which is tyrosine or phenylalanine;    -   R₂ is cysteine or pencillamine;    -   R₃ is 0 amino acids;    -   R₄ is 7-8 amino acids;    -   R₅ is 0 amino acids;    -   R₆ is cysteine or pencillamine;    -   R₇ is 1-5 amino acid residues, at least one of        which is tyrosine or phenylalanine;        wherein:    -   R₁, R₂, R₃, R₄, R₅, R₆ and R₇, taken together, are 30 amino        acids or less, and wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇ are        otherwise as set forth above.

In certain embodiments the peptide is B2-S23-BPT:P-C-P-I-N-C-T-H-S-C-V-D-L-D-D-K-G-C-P-A-E-Q-R-A-S-P-L-T-S-I (SEQ ID NO:38).

In some preferred embodiments of generating antibodies against IgSFmembers, the peptide is:C-K-V-E-L-M-Y-P-P-P-Y-F-V-G-M-G-N-G-T-Q-I-Y-V-I-D-P-E-P-C (SEQ ID NO:36).

In some preferred embodiments of generating antibodies against IgSFmembers, the peptide is:C-K-I-E-F-M-Y-P-P-P-Y-L-D-N-E-R-S-N-G-T-I-I-H-I-K-E-K-H-L-C (SEQ ID NO:29).

In some preferred embodiments the peptide is:C-S-L-S-I-F-D-P-P-P-F-Q-E-R-N-L-S-G-G-Y-L-H-I-Y-E-S-Q-L-C (SEQ ID NO:30).

In some preferred embodiments the peptide is B2-S22-APE:Y-C-P-I-W-K-F-β-D-E-E-C-Y (SEQ ID NO: 31).

In some preferred embodiments the peptide is B1-S22-ALG:Y-C-L-V-W-K-Y-A-D-A-G-C-Y (SEQ ID NO: 32).

In some preferred embodiments the peptide is B3-S22-APQ:Y-C-P-I-Y-K-Y-P-D-V-Q-C-Y (SEQ ID NO: 33).

In some preferred embodiments the peptide is B4-S22-AFD:Y-C-F-I-F-K-Y-A-D-P-D-C-Y (SEQ ID NO: 34).

In some preferred embodiments the peptide is B2-S22-AFA:Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO: 35).

Those having ordinary skill in the art can readily construct moleculesaccording to the above formulae and determine whether or not thecompounds are active as erbB binding compounds which prevent andeliminate the erbB-mediated transformation phenotype.

The peptides of the invention may be dimerized with each other to formhomodimers or with other compounds including compounds of the inventionto form heterodimers. In preferred dimers, the residues involved in thechemical bound which links the monomers is in the R₁ position of thecompounds of the invention.

Mimetics of the peptides which mimic erbB, TNF, or IgSF receptors may beproduced. Such mimetics may be tested in the assays set forth in theExamples.

According to the present invention, peptides and mimetics that mimicsites on erbB, TNF, or IgSF receptors are useful to prevent dimerizationof receptors and thereby down modulate the kinase activity of thereceptors. When bound, the peptides and mimetics eliminate or reducetyrosine kinase activity and/or receptor signaling that results in anelimination or reduction in cell proliferation levels and anon-transformed, quiescent phenotype. The peptides and mimetics aretherefore useful in the treatment of individuals suspected of havingerbB tumors or TNF and IgSF-mediated pathologies, and in the preventionof such erbB tumor formation. The cells in the individuals that wouldturn into tumors in an untreated individual do not become fullytransformed and do not become tumors in individuals treated by themethods. When administered to individuals who have been identified asbeing susceptible to or otherwise at risk of developing tumors, thepeptides and mimetics may induce antibodies that bind to erbB or TNFmonomers, thereby preventing the elevation in tyrosine kinase activityor signaling associated with oligomerization of the receptors. Thetyrosine kinase activity in the cell may never become sufficientlyelevated and the cell remains non-transformed.

Peptides and mimetics of erbB receptors, TNF or IgSF family receptorsare useful in anti-tumor compositions and can be produced by thoseskilled in the art using readily available starting materials. U.S. Pat.No. 5,637,677 and its parent applications thereof disclose detailedguidance on the production of mimetics.

According to preferred embodiments, a peptide or mimetic is designedbased on a known region of an erbB, TNF, or IgSF receptor. In somepreferred embodiments, the peptide or mimetic mimics an extracellulardomain of an erbB, TNF, or IgSF receptor. In some more preferredembodiments, the peptide or mimetic mimics the second cysteine richdomain proximal to the transmembrane domain (S22 loop). In somepreferred embodiments, the peptide mimetic mimics of the S23 loop.According to some embodiments, the peptide mimetics of the presentinvention mimic cystine knots comprising regions.

In some preferred embodiments, the peptides and/or mimetics areexocyclic. In some preferred embodiments, the peptides and/or mimeticsmimic full cystine knots. In some embodiments, the peptides and/ormimetics mimic a portion of a cystine knot.

In addition, peptides and/or mimetics may mimic assembly or functionalepitopes of erbB2 and erbB1, erbB1 and erbB3, erbB1 and erbB4, erbB2 anderbB3, erbB2 and erbB4, erbB3 and erbB4, assembly or functional epitopesof TNF receptors, or assembly and functional epitopes of IgSF members.

In some embodiments, peptides and/or mimetics are provided which mimicdimerization sites of erbB, TNF, or IgSF receptors, and may induceantibodies. In a preferred embodiment, the peptides and/or mimeticsmimic a dimerization site of erbB1 or erbB2. Binding of the peptidesand/or mimetics or induced antibodies to the dimerization site preventsthe dimerization of the receptor, and, preferably, reduces or eliminatesreceptor signaling.

In some embodiments, peptides and/or mimetics are provided which mimicconformation sites of erbB, TNF, or IgSF receptors, and may induceantibodies. In a preferred embodiment, the peptides and/or mimeticsmimic a conformation site of erbB1 or erbB2. Binding of the peptidesand/or mimetics or induced antibodies to the conformation site changesthe conformation of the receptor and prevents the dimerization of thereceptor, and, preferably, reduces or eliminates receptor signaling.

The peptides and/or mimetics of the invention are useful in thetreatment of erbB tumors either as a component of a compositionadministered to a patient, as a component of a composition administeredto a patient in combination with radiation therapy and/or chemotherapy,or as a component of a composition administered to a patient thatcomprises the peptides and/or mimetics conjugated to a radioactive orchemotherapeutic agent.

The peptides and/or mimetics of the invention are useful in theprevention of erbB tumors or TNF- or IgSF-mediated pathologies either asa component of a composition administered to a patient, as a componentof a composition administered to a patient in combination with radiationtherapy and/or chemotherapy, or as a component of a compositionadministered to a patient that comprises the peptides and/or mimeticsconjugated to a radioactive or chemotherapeutic agent.

The peptides and/or mimetics of the invention are useful for raisingantibodies. In some embodiments, an antibody is produced by immunizing asuitable host with a peptide selected from the group consisting ofY-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO:3);C-K-V-E-L-M-Y-P-P-P-Y-F-V-G-M-G-N-G-T-Q-I-Y-V-I-D-P-E-P-C (SEQ ID NO:28); C-K-I-E-F-M-Y-P-P-P-Y-L-D-N-E-R-S-N-G-T-Q-I-H-I-K-E-K-H-L-C (SEQ IDNO: 29); C-S-L-S-I-F-D-P-P-P-F-Q-E-R-N-L-S-G-G-Y-L-H-I-Y-E-S-Q-L-C (SEQID NO: 30); B2-S22-APE: Y-C-P-I-W-K-F-P-D-E-E-C-Y (SEQ ID NO: 31);B1-S22-ALG: Y-C-L-V-W-K-Y-A-D-A-G-C-Y (SEQ ID NO: 32); B3-S22-APQ:Y-C-P-I-Y-K-Y-P-D-V-Q-C-Y (SEQ ID NO: 33); B4-S22-AFD:Y-C-F-I-F-K-Y-A-D-P-D-C-Y (SEQ ID NO: 34); B2-S22-AFA:Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO: 35). In some embodiments the antibodybinds to a cysteine rich domain of an erbB receptor. In someembodiments, the peptides or mimetics induce antibodies that bind tofunctional sites of erbB, TNF, or IgSF family receptors.

The present invention also contemplates antibodies produced byimmunizing a suitable host with a peptide selected from the groupconsisting of SEQ ID NOS:1-37 as well as antibodies produced byimmunizing a suitable host with a peptide having the sequence in reverseorder selected from the group consisting of SEQ ID NOS:1-37.

Antibodies

The present invention describes antibodies against receptor assemblydomains.

According to certain embodiments, antibodies that bind to, for example,either erbB1, erbB2, erbB3 or erbB4 and are useful to preventoligomerization-mediated signaling of receptors and thereby downmodulate kinase activity of the receptors. When bound, the antibodieseliminate or reduce tyrosine kinase activity that results in anelimination or reduction in cell proliferation levels and anon-transformed, quiescent phenotype. The antibodies are thereforeuseful in the treatment of individuals suspected of having erbB tumorsand in the prevention of such tumor formation. The cells in theindividuals under treatment (that would otherwise turn into tumors in anuntreated individual) do not become transformed and do not becometumors. When administered to individuals who have been identified asbeing susceptible to or otherwise at risk of developing tumors, theantibodies bind to, for example, erbB1, erbB2, erbB3 or erbB4, therebypreventing the elevation in tyrosine kinase activity associated withdimerization of the receptors. The tyrosine kinase activity in the cellnever becomes sufficiently elevated and the cell remainsnon-transformed.

Antibodies useful in anti-tumor compositions can be produced by thoseskilled in the art using readily available starting materials. Thetechniques for producing monoclonal antibodies are outlined in Harlow,E. and D. Lane, (1988) ANTIBODIES: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor N.Y., which provides detailedguidance for the production of hybridomas and monoclonal antibodieswhich specifically bind to target proteins. Briefly, the protein ofinterest, rodent or human erbB2, for example, or cells which expressthis protein, are injected into mice. The mouse is then boosted with theprotein. The spleen of the mouse is removed and the spleen cells areisolated and fused with immortalized mouse cells. The hybrid cells, orhybridomas, are cultured and those cells which secrete antibodies areselected. The antibodies are analyzed and, if found to specifically bindto the protein of interest, the hybridoma which produces them iscultured to produce a continuous supply of antigen specific antibodies.Humainized or camelized antibodies may be generated using techniqueswell-known to those skilled in the art.

In some embodiments, antibodies are induced by immunizing an erbB or TNFreceptor, or portion thereof. In more preferred embodiments, antibodiesare induced by immunizing with a cystine knot peptide mimetic or peptidemimetic from cysteine rich domains with or without constraints.

In certain embodiments of the invention, the complementarity determiningregion of antibodies is formed from immunoglobulin heavy chains only,i.e., without immunoglobulin light chains. Such antibodies may bederived, for example, using antibody heavy chains from camelids, camelsor llamas. (Davies et al., (1996) Protein Engineering, 9, 531-537;Reichmann, L., (1996) J. Mol. Biol., 259, 957-969; Davies et al., (1995)Biotechnology, 13, 475-479; Decanniere et al., (1999) Structure FoldDes., 7, 361-370). The structure of, e.g., the camelid single domainantibody is useful as a scaffold for anti-idiotypic vaccinations andexpression of peptidomimetics (Muyldermans et al., (1999) J. Mol.Recognit., 12, 131-140; Dumoulin, et al., (2002), Prot. Sci., 11,500-515; Tanha et al., (2001), J. Biol. Chem., 276, 24774-24780.)

In some embodiments, an antibody is produced by immunizing a suitablehost with a peptide selected from the group consisting of:Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO:3);C-K-V-E-L-M-Y-P-P-P-Y-F-V-G-M-G-N-G-T-Q-I-Y-V-I-D-P-E-P-C (SEQ ID NO:28); C-K-I-E-F-M-Y-P-P-P-Y-L-D-N-E-R-S-N-G-T-I-I-H-I-K-E-K-H-L-C (SEQ IDNO:29); C-S-L-S-I-F-D-P-P-P-F-Q-E-R-N-L-S-G-G-Y-L-H-I-Y-E-S-Q-L-C (SEQID NO: 30); B2-S22-APE: Y-C-P-I-W-K-F-P-D-E-E-C-Y (SEQ ID NO: 31);B1-S22-ALG: Y-C-L-V-W-K-Y-A-D-A-G-C-Y (SEQ ID NO: 32); B3-S22-APQ:Y-C-P-I-Y-K-Y-P-D-V-Q-C-Y (SEQ ID NO: 33); B4-S22-AFD:Y-C-F-I-F-K-Y-A-D-P-D-C-Y (SEQ ID NO: 34); B2-S22-AFA:Y-C-F-P-D-E-E-G-A-C-Y (SEQ ID NO: 35). In some embodiments the antibodybinds to a cystine knot of an erbB receptor.

According to some embodiments of the invention, a monoclonal antibody isprovided that binds to human/rat erbB1, erbB2, erbB3, orerbB4-receptors.

Preferably, the binding affinity for antigens is at least 1×10⁶ Ka. morepreferably 1×10⁷ Ka, more preferably 2×10⁷ Ka, more preferably 1×10⁸ Ka.

According to preferred embodiments of the invention, a monoclonalantibody is provided that binds to a human/rat TNF-receptor.

Preferably, the binding affinity for TNF antigens is at least 1×10⁶ Ka.Preferably, the binding affinity for TNF receptors is at least 5×10⁶ Ka,more preferably 1×10⁷ Ka, more preferably 2×10⁷ Ka, more preferably1×10⁸ Ka.

According to preferred embodiments of the invention, a monoclonalantibody is provided that binds to a human/rat IgSF family-receptor.

Preferably, the binding affinity for IgSF family-antigens is at least1×10⁶ Ka. Preferably, the binding affinity for IgSF family-receptors isat least 5×10⁶ Ka, more preferably 1×10⁷ Ka, more preferably 2×10⁷ Ka,more preferably 1×10⁸ Ka.

The present invention is useful to therapeutically treat an individualidentified as suffering from erbB tumors and/or TNF of IgSF pathologies.The present invention is also useful to prophylactically treat anindividual who is predisposed to develop erbB tumors or TNF/IgSF-relatedpathologies or who has had erbB tumors or TNF/IgSF-related pathologiesand is therefore susceptible to a relapse or recurrence. The presentinvention is also useful, inter alia, to image erbB tumors orTNF/IgSF-related pathologies and otherwise detect them.

The antibodies of the invention are useful in the treatment of erbBtumors or TNF/IgSF-related pathologies either as a component of acomposition administered to a patient, as a component of a compositionadministered to a patient in combination with radiation therapy and/orchemotherapy, or as a component of a composition administered to apatient that comprises the antibody conjugated to a radioactive orchemotherapeutic agent.

The antibodies of the invention are useful in the prevention of erbBtumors or TNF/IgSF-related pathologies either as a component of acomposition administered to a patient, as a component of a compositionadministered to a patient in combination with radiation therapy and/orchemotherapy, or as a component of a composition administered to apatient that comprises the antibody conjugated to a radioactive orchemotherapeutic agent.

The antibodies of the invention are useful in the imaging of erbB tumorsor TNF/IgSF-related pathologies as, for example, a detectable componentof a composition administered to a patient.

General Features of Receptors with a Cysteine Rich Domain (CRD)

The crystal structure of the TNF receptor, both in complexed anduncomplexed forms, provides a general picture of how this receptorfamily binds to their ligands (Banner, et al. 1993; Eck and Sprang 1989;Eck, et al. 1992) and of associated ligand induced conformationalchanges. The receptors associate by structural elements known ascysteine-knots. The cystine knot in the TNF receptor family consists of42 amino acid residues with 6 cysteine residues forming 3 inter-chaindisulfide bonds to create the structural motif. The three-dimensionalstructure reveals the cysteine-knot repeats of about 30 Å in length arearranged in a head-to-tail fashion exposing the loops on one side of thereceptor. These cysteine-knots are involved in both oligomerization andligand binding (Eck and Sprang 1989; Eck, et al. 1992). Uncomplexed TNFreceptors are observed as dimers. In the dimeric form, the first andlast cysteine domains involve dimeric contacts (Naismith, et al. 1995).It has been argued that the TNF dimeric receptor forms regulate thefunction of the trimeric forms (Naismith, et al. 1995).

ErbB receptors share significant homology with TNF receptor (Ward, etal. 1995) in the cysteine rich domains. The recently determined crystalstructure of insulin receptor (Garrett, et al. 1998b) confirmed thatreceptors with cysteine rich domains (CRD) adopt a similar conformation,namely the “cystine knot” observed in TNF receptors.

Anti-HER2/p185^(c-neu) Antibody Binds to the Ectodomain and Leads top185 Internalization

Disabling a protein responsible for maintenance of the malignantphenotype reverses transformation. This body of work (Drebin, et al.1986, Symp Fundam Cancer Res 38:277-289; Drebin, et al. 1986, Proc NatlAcad Sci USA 83:9129-9133) was subsequently substantiated (Carter, etal. 1992) and has now has been approved for clinical use as “HERCEPTIN”(Baselga, et al. 1998; Pegram, et al. 1998).

Antibodies to the ectodomain of p185 can reverse the phenotype oftransformed cells by leading to the rapid downmodulation of the receptorfrom the cell surface (Drebin, et al. 1986, Proc Natl Acad Sci USA83:9129-9133). The removal of the transforming receptor from the cellsurface in vitro was associated with a reduction in the malignantphenotype and a conversion of the cell phenotype into a more normal oneas judged by cell growth, phenotype, and growth in soft agar. Theanti-receptor antibody p185 complex was visualized and shown to enterthe cell and lead to p185 degradation (Brown, et al. 1994). Subsequentin vivo studies showed that the administration of anti-receptorantibodies alone could cause retardation of tumor growth.

Studies in small animals which had been treated to eliminate complementor macrophages clearly indicated that the effect of the antibodies waspredominantly but not entirely direct and was related to receptordownmodulation (Drebin, et al. 1988).

Biochemical Aspects of Creating Mimetics that are Used In Vitro asInhibitors

Reducing a macromolecule to a small molecule with similar function is ageneral chemical problem. There have been some attempts to designmini-proteins by transplanting functional units onto suitable scaffolds(Vita, et al. 1998) and minimizing antibodies to single chain antibodies(Magliani, et al. 1997).

It is now established that conformation constrained peptides are morebioactive than unconstrained ones. In recent years, the chemistry ofpeptide cyclization by solid phase synthesis and other methods hasimproved dramatically (Burgess, et al. 1996; Goodman and Shao 1996;Hanessian, et al. 1997; Koskinen and Hassila 1996; Kuhn, et al. 1997;Waid, et al. 1996; Zuckermann 1993) providing more opportunity todevelop peptides into peptidomimetics.

General principles to create cyclic peptide mimetics that have beenadopted and in some cases (such as eptifitimide (Integrilin) aglycoprotein 11b 111a inhibitor (COR Therapeutics)) have becomeclinically available. Aromatic residues placed at the termini ofcyclically constrained small peptides increases the activity of mimetics(Takasaki, et al. 1997; Zhang, et al. 1996). Employing suchmodifications has allowed creation of high affinity mimetics of antibodymimics based on CDRs (Park, et al. 2000), CD4 (Zhang, et al. 1997), IL4receptor loop mimetics, anti-CD3 antibody mimics, and TNF cystine knotmimetic that affect TNFα binding to its receptor (Takasaki, et al.1997).

Cyclic Peptidomimetics are Superior Immunogens

Antigenic sites recognized by antibody consist of discretethree-dimensional surfaces (Van Regenmortel 1989; Van Regenmortel 1996).Spatial distribution of antigenic residues on the surface of proteinsare often reproduced by constrained peptides (Nayak, et al. 1998;Posthumus, et al. 1991; Valero, et al. 1995; van der Werf, et al. 1994).An example involves the foot-mouth- and disease virus. A dominantantigenic site consists of flexible loop and immunization with aconstrained peptide of this loop elicits higher affinity andneutralizing antibodies than the MAb elicited by linear peptides (Patel,et al. 1999; Valero, et al. 1995). These studies suggest that rigid, butspatial mimics of antigenic regions can be useful as immunogens and maybe used to induce MAb to erb receptor interaction surfaces. These MAbare useful as in treatment of erbB pathologies.

Therapeutic Methods

The present invention is useful to therapeutically treat an individualidentified as suffering from erbB tumors and/or TNF or IgSF-relatedpathologies in order to reverse the transformed phenotype of the tumorcells and/or induce tumor cell death. The present invention is useful toprophylactically treat an individual who is predisposed to develop erbBtumors and/or TNF or IgSF-related pathologies or who has had erbB tumorsand/or TNF or IgSF-related pathologies and is therefore susceptible to arelapse or recurrence.

When a therapeutically effective amount of an antibody, peptide ormimetic of the present invention is administered to an individual whohas erbB cancer, the proliferation rate of tumor cells is slowed down oreliminated.

Prophylactic methods are useful to treat an individual who ispredisposed to develop erbB tumors and/or TNF or IgSF-relatedpathologies or who has had erbB tumors and/or TNF or IgSF-relatedpathologies and is therefore susceptible to a relapse or recurrence.

In some embodiments, the methods relate to treating patients sufferingfrom human adenocarcinomas such as gastric, lung and pancreaticadenocarcinomas and human breast and ovarian carcinomas as well as humanprostate cancer. In some embodiments, the methods relate to treatingpatients suffering from glial tumor progression, particularly inglioblastoma, the most malignant glial tumor. In some embodiments, themethods relate to treating patients suffering from human epithelialmalignancies erythroid leukemia, fibrosarcoma, angiosarcoma andmelanoma. In some embodiments the present invention provides methods oftreating such diseases/disorders comprising the step of diagnosing apatient a suffering from a multimer-associated disease/disorder and thentreating the disease/disorder in accordance with other methods of theinvention.

Radiation therapy may commence anytime after a sufficient amount of timehas elapsed for the antibodies or peptide mimetics to bind to thereceptors. Generally, the individual is exposed to radiation in somecases 1-10 minutes after, in some cases 1-10 hours after, and in somecases up to 24-72 hours after administration of the antibodies, peptidesor mimetics. In some cases, the radiation is provided in a single dosewhile in some embodiments, multiple doses are administered over severalhours, days and/or weeks. The antibodies render the radiation resistanttumor cells radiation sensitive. Gamma radiation is delivered accordingto standard radiotherapeutic protocols using standard dosages andregimens. The administration of the antibodies, peptides or mimeticsrenders the radiation more effective in eradicating tumor cells.

The individual may be treated with antibodies, peptides or mimetics incombination with a cytotoxic chemotherapeutic agent in addition to or inlieu of exposure to a therapeutic amount of gamma radiation.Chemotherapy may commence anytime before or after the antibodies orpeptide mimetics are administered, or with the antibodies, peptides ormimetics themselves. Generally, the individual is administered thechemotherapeutic in some cases 1-10 minutes after, in some cases 1-10hours after, and in some cases up to 24-72 hours after administration ofthe antibodies, peptides or mimetics. In some cases, thechemotherapeutic is provided in a single dose while in some embodiments,multiple doses are administered over several hours, days and/or weeks.The antibodies render the tumor cells more sensitive to cytotoxicagents. Chemotherapeutics are delivered according to standardradiotherapeutic protocols using standard agents, dosages and regimens.In some embodiments, the chemotherapeutic is selected from the groupconsisting of cisplatin, doxirubicin, danurubicin, tamoxiphen, taxol,and methotrexate. In some embodiments, the individual is treated withantibodies and/or peptides and/or mimetics of the present invention incombination with two or more chemotherapeutics, each administered priorto, simultaneous with, or after the other chemotherapeutics. In someembodiments, chemotherapy and radiation treatments are both employedfollowing the administration of the active agent. In such embodiments,standard combinations of two or more therapeutic modalities are used inconjunction with administration of the antibodies and/or peptides and/ormimetics.

According to some embodiments of the invention, the patient is treatedwith radiation and/or other chemotherapy in conjunction with theadministration of pharmaceutical compositions according to theinvention. Chemotherapy approaches include administration of cytotoxicand or cytostatic agents. It has been observed that expression ofnucleotide molecules according to the invention in erbB-associatedtumors renders the tumors radiosensitized. That is, the tumors are morevulnerable to destruction by radiation during radiotherapy when thepatient is treated with pharmaceutical compositions according to theinvention. The use of multiple therapeutic approaches provides thepatient with a broader based intervention. In some preferredembodiments, treatment with pharmaceutical compositions according to thepresent invention is preceded by surgical intervention. In preferredembodiments, radiotherapy follows administration of pharmaceuticalcompositions according to the invention. In preferred embodiments, theradiation therapy using gamma radiation is provided followingadministration of compositions which convert radiation resistant tumorsinto radiation sensitive tumors. Those skilled in the art can readilyformulate an appropriate radiotherapeutic regimen. Carlos A Perez &Luther W Brady: Principles and Practice of Radiation Oncology, 2nd Ed.JB Lippincott Co, Phila., 1992, which is incorporated herein byreference describes radiation therapy protocols and parameters which canbe used in the present invention. For GBMs (glioblastoma, the mostmalignant glial brain tumor), Simpson W. J. et al.: Influence oflocation and extent of surgical resection on survival of patients withglioblastoma multiforms: Results of three consecutive Radiation TherapyOncology Group (RTOG) clinical trials. Int J Radiat Oncol Biol Phys26:239-244, 1993, which is incorporated herein by reference, describesclinical protocols useful in the methods of the present invention.Similarly, for brain tumors, see Borgelt et al., The palliation of brainmetastases: Final results of the first two studies of the RadiationTherapy Oncology Group. Int J Radiat Oncol Biol Phys 6:1-9, 1980, whichis incorporated herein by reference and describes clinical protocolsuseful in the methods of the present invention.

The antibodies, peptides or mimetics of the present invention may beused to prevent tumors in individuals susceptible to such tumors.According to one aspect of the invention, antibodies are administeredprophylactically to individuals susceptible to developing erbB tumors.According to another aspect of the invention, peptides are administeredprophylactically to individuals susceptible to developing erbB tumorsand/or TNF or IgSF-related pathologies. According to another aspect ofthe invention, mimetics are administered prophylactically to individualssusceptible to developing erbB tumors and/or TNF or IgSF-relatedpathologies. Those having ordinary skill in the art can readilydetermine whether an individual may be susceptible to such tumors. Themethods are particularly useful in high-risk individuals who, forexample, have a family history of erbB cancer, or show a geneticpredisposition. Additionally, the methods are particularly useful toprevent patients from having recurrences of erbB tumors who have haderbB tumors removed by surgical resection or who have been diagnosed ashaving erbB-cancer in remission. In some preferred embodiments, thecancer is erbB2/erbB1 cancer.

Methods of treatment comprise administering single or multiple doses ofthe antibodies, peptides or mimetics of the present invention. Preferredfor human pharmaceutical use are injectable pharmaceutical compositionsthat are sterile, pyrogen free and comprise the antibodies, peptides ormimetics in combination with a pharmaceutically acceptable carrier ordiluent.

The antibodies, peptides or mimetics of the present invention may beused to treat individuals suffering from erbB tumors. According to oneaspect of the invention, antibodies are administered to individualssuspected of having such tumors. According to another aspect of theinvention, peptide mimetics are administered to individuals suspected ofhaving such tumors. Those having ordinary skill in the art can readilydetermine whether an individual may have a tumor likely to be an erbBtumor. Biopsy protocols can be performed to identify tumor samples anddetermine whether or not they are erbB tumors. According to someaspects, the patient is treated with the antibodies, peptides ormimetics in conjunction with chemotherapy and/or radiation therapy. Forexample, following administration of the antibodies, peptides ormimetics, the patient may be treated with a therapeutically effectiveamount of anti-cancer radiation such as gamma radiation. Moreover, someembodiments provide chemotherapeutic treatment in combination with theantibodies, peptides or mimetics. Other aspects of the invention involvethe administration of antibodies, peptides or mimetics in conjunctionwith chemotherapy and/or radiation therapy. In some preferredembodiments, the cancer is erbB2/erbB1 cancer.

The present invention is also useful for preventing erbB tumors orformation of a vaccine for prevention or treatment of erbB tumors. Theterm “vaccine”, as used herein, broadly refers to any compositions thatmay be administered to an organism to protect the organism against aninfectious disease. The term “protect”, as used herein to describevaccines, means prevention or treatment of the infectious disease.Vaccines protect against diseases, e.g., erbB tumors, by inducing orincreasing an immune response in an organism against infectious agentsor against cells that have undergone transition to an abnormal ordiseased state. An erbB vaccine, e.g., illicits an immune responseagainst cells that express abnormal amounts or forms of an erbB receptorsuch as that found on abnormal or transformed cells.

A vaccine generally comprises a therapeutically effective dose of animmunogen (e.g., an antigen) and, preferably, an adjuvant and/or apharmaceutically acceptable carrier.

A vaccine may be administered to an organism, e.g., by inhalation orinsufflation (either through the mouth or the nose), or by oral, buccal,rectal or parenteral administration (e.g., by subcutaneous,intramuscular, intraorbital, intracapsular, intraspinal, intrasternal orintravenous injection and the like). A vaccine may also be administeredby particle-mediated transfer (e.g., using a “particle gun”). See forexample, Gainer et al., J. Neurooncol 2000, 47:23-30; Koide et al., JpnJ. Pharmacol 2000, 83:167-174; Kuriyama et al., Gene Ther. 2000,7:1132-1136; and Yamauchi et al., J. Exp. Zool. 2000, 287:285-293. Suchparticle transfer methods are particularly preferred for DNA or vectorvaccines (discussed below), e.g., using a “gene gun”.

A vaccine of the invention may comprise, for example, a polypeptidevaccine or a DNA vaccine. The term “polypeptide vaccine” refers to avaccine comprising an immunogenic polypeptide, which may be an antigen,and therefore activates an immune response in an organism. The term “DNAvaccine” is an informal term of art, and is used herein to refer tovaccines delivered by means of a recombinant vector. An alternative termused herein is “vector vaccine” (since some potential vectors, forexample retroviruses and lentiviruses, are RNA viruses and since in someinstances non-viral RNA instead of DNA may be delivered to cells).

The peptides and mimetics disclosed herein may be used in a vaccine. Incertain embodiments, the vaccine comprises a subdomain W sequence of anerbB receptor. In a preferred embodiment, the erbB receptor subdomain IVsequence is from human erbB. Most preferably the subdomain W sequencehas a sequence selected from the group consisting of SEQ ID: 39, SEQ ID:40, SEQ ID: 41 and SEQ ID: 42. In other embodiments, the vaccinecomprises a peptide of between 10-25 contiguous amino of an erbBreceptor subdomain IV. In another embodiment, the vaccine comprises anerbB subdomain IV wherein between 1-10 amino amino acids of thesubdomain have been substituted with a conservative amino acid. Suchconservative amino acid changes are well known to those in the art.

It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, W H Freeman and Co.: 1981).

Methods of Imaging and Diagnosing Mammalian Tumors

The present invention is also useful, inter alia, to image erbB tumorsand TNF or IgSF-related pathologies and otherwise detect or diagnosethem.

The antibodies, peptides or mimetics of the present invention can belabeled or otherwise made detectable. For example, a detectable antibodyis useful as an imaging agent and reagent in diagnostic procedures thatare used to identify tumors. Labeled antibodies can be administered toindividuals suspected of suffering from erbB tumor and/or TNF orIgSF-related pathologies. The labeled antibodies will bind to the highdensity of receptors on cells and thereby accumulate on the tumor cells.Using standard imaging techniques, the site of the tumors can bedetected.

One aspect of the invention therefore relates to methods of imagingtumors. Such methods comprise the steps of administering a detectableantibody, peptide or mimetic to an individual who is suffering from oris susceptible to erbB cancer and detecting the location of thedetectable antibody, peptide or mimetic within the body of theindividual or within a sample obtained from said individual.

The antibodies, peptides or mimetics bind to receptors present on cellsurfaces and are therefore useful as diagnostic/characterizing reagentsin diagnostic kits. When a tissue sample from an individual is contactedwith an antibody, peptide or mimetic, the antibody, peptide or mimeticwill bind to the receptors present on cells. Labeled antibodies,peptides or mimetics are also useful as in vitro reagents to quantifythe amount of receptors present in the cell. Such information indicateswhether or not a tumor is erbB-related and, therefore, whether specifictreatments should be used or avoided. Using standard techniques, samplesbelieved to include tumor cells are obtained and contacted with labeledantibodies, peptides or mimetics. After removing any unbound, labeledantibodies, peptides or mimetics, the quantity of labeled antibodies,peptides or mimetics bound to the cell, or the quantity of antibodies,peptides or mimetics removed as unbound, labeled antibodies isdetermined. The information directly relates to the amount of receptors.This information is useful in formulating the prognosis and course oftreatment to be imposed on the individual.

Imaging agents are useful in diagnostic procedures as well as inprocedures used to identify the location of tumors. Imaging can beperformed by many procedures well-known to those having ordinary skillin the art and the appropriate imaging agent useful in such proceduresmay be conjugated to antibodies by well-known means. Imaging can beperformed, for example, by radioscintigraphy, nuclear magnetic resonanceimaging (MRI) or computed tomography (CT scan). The most commonlyemployed radiolabels for imaging agents include radioactive iodine andindium. Imaging by CT scan may employ a heavy metal such as an ironchelate. MRI scanning may employ chelates of gadolinium or manganese.Additionally, positron emission tomography (PET) may be possible usingpositron emitters of oxygen, nitrogen, iron, carbon, or gallium.

In another embodiment, a diagnostic method is provide in whichradiolabeled F(ab)' fragments prepared from the monoclonal antibodies ofthe present invention are administered to patients. The location andsize of the tumor are determined by gamma-scintigraphy to detect theradiolabeled F(ab□) fragments.

In some embodiments, tumors can be diagnosed by contacting tissueportions of the tumor with a antibody being labeled with an indicator.The antibody binds to the erbB oligomer present in the cells of thetissue portion. The indicator is then detected. In preferred embodimentsof the invention, the indicator comprises biotinylated horse anti-mouseimmunoglobulin and streptavidin-biotinylated-peroxidase. The indicatorin detected by contacting the indicator with a chromogenic substratewhich preferably comprises 3,3′-diaminobenzidine, hydrogen peroxide andimidazole. The chromogenic substrate is then detected by microscopy.

In some embodiments, tumors can be diagnosed by contacting tissueportions of the tumor with a labeled peptide or mimetic. The labeledpeptide or mimetic binds to the erbB receptor present in the cells ofthe tissue portion. The indicator is then detected. In preferredembodiments of the invention, the indicator comprises biotinylated horseanti-mouse immunoglobulin and streptavidin-biotinylated-peroxidase. Theindicator in detected by contacting the indicator with a chromogenicsubstrate which preferably comprises 3,3′-diaminobenzidine, hydrogenperoxide and imidazole. The chromogenic substrate is then detected bymicroscopy.

Pharmaceutical Compositions

The invention further provides an injectable composition for treatmentof a mammalian cancer tumor having cells that express erbB receptors orTNF receptors on the surfaces of the cells. In accordance with theinvention, the composition comprises an antibody, peptide or mimeticspecific to the shared epitope and a pharmaceutically acceptableinjection vehicle.

When a therapeutically effective amount of an antibody, peptide ormimetic of the present invention is administered to an individual whohas erbB cancer or TNF or IgSF-related pathology, the proliferation rateof cells is slowed down or eliminated.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the antibodies, peptides ormimetics to reach the agent's site of action in the body of a mammal.Because proteins are subject to being digested when administered orally,parenteral administration, i.e., intravenous, subcutaneous,intramuscular, would ordinarily be used to optimize absorption.Formulations may be devised which protect the antibodies, peptides ormimetics and render them resistant to many proteases, thus making themorally available.

In addition to pharmaceutical compositions which comprise antibodies,peptides or mimetics alone or in combination with other cancertherapeutics, therapeutic and diagnostic pharmaceutical compositions.The pharmaceutical compositions which comprise conjugated compositionsmay be used to diagnose or treat individuals suffering from erbB and/orTNF cancer.

Pharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

The compositions may include additional components to render them moreeffective. For example, a composition of the invention may comprisemultiple anti-p185 antibodies. The compositions may include otheranti-cancer agents such as, for example, cis-platin, methotrexate,and/or GM-CSF. Such compositions would be particularly useful foradministration to patients diagnosed and treated for erbB-associatedcancer.

Generally, additives for isotonicity can include sodium chloride,dextrose, mannitol, sorbitol and lactose. In some cases, isotonicsolutions such as phosphate buffered saline are used. Stabilizersinclude gelatin and albumin. In some embodiments, a vasoconstrictionagent is added to the formulation. The pharmaceutical preparationsaccording to the present invention are preferably provided sterile andpyrogen free.

One of skill in the art of pharmaceutical formulations, e.g., having anadvanced degree in Pharmaceutics or Pharmaceutical Sciences, can preparea variety of appropriate dosage forms and formulations for thecompositions of the invention with no more than routine experimentation.A number of texts in the field, e.g., Remington's PharmaceuticalSciences and The U.S. Pharmacopoeia/National Formulary, latest editions,provide considerable guidance in this respect, each of which isincorporated by reference in its entirety

A pharmaceutically acceptable formulation will provide the activeingredient(s) in proper physical form together with such excipients,diluents, stabilizers, preservatives and other ingredients as areappropriate to the nature and composition of the dosage form and theproperties of the drug ingredient(s) in the formulation environment anddrug delivery system.

Kits

Kits of the invention comprise detectable antibodies and/or peptidesand/or mimetics and instructions for performing assays of the invention.Optionally, kits may also contain one or more of the following:containers which comprise positive controls, containers which comprisenegative controls, photographs of representative examples of positiveresults and photographs of representative examples of negative results.

Conjugates

Antibodies, peptides or mimetics may be conjugated to a detectableand/or cytotoxic agent. In conjugated compositions, the antibodies,peptides or mimetics of the invention deliver the active agent to cells.Thus, cells with the receptors will be contacted with more active agentsthan other cells. The active agent is useful to image, inhibitproliferation of and/or kill the cell. The active agent may be atherapeutic agent or an imaging agent.

Some chemotherapeutic agents may be used as active agents and conjugatedwith antibodies, peptides or mimetics. Chemotherapeutics are typicallysmall chemical entities produced by chemical synthesis and includecytotoxic drugs, cytostatic drugs as well as antibodies which affectcells in other ways such as reversal of the transformed state to adifferentiated state or those which inhibit cell replication. Examplesof chemotherapeutics include, but are not limited to: methotrexate(amethopterin), doxorubicin (adrimycin), daunorubicin,cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan,chlorambucil, and other nitrogen mustards (e.g. cyclophosphamide),cis-platinum, vindesine (and other vinca alkaloids), mitomycin andbleomycin.

Active agents may be toxins: complex toxic products of various organismsincluding bacteria, plants, etc. Examples of toxins include but are notlimited to: ricin, ricin A chain (ricin toxin), Pseudomonas exotoxin(PE), diphtheria toxin (DT), Clostridium perfringens phospholipase C(PLC), bovine pancreatic ribonuclease (BPR), pokeweed antiviral protein(PAP), abrin, abrin A chain (abrin toxin), cobra venom factor (CVF),gelonin (GEL), saporin (SAP), modeccin, viscumin and volkensin. Proteintoxins may be produced using recombinant DNA techniques as fusionproteins that include peptides of the invention. Protein toxins may alsobe conjugated to antibodies by non-peptidyl bonds.

Radioisotopes may be conjugated to antibodies, peptides or mimetics toprovide compositions that are useful as therapeutic agents or forimaging procedures. Examples of radioisotopes which useful in radiationtherapy include: ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re,¹⁹⁹Au, ²¹¹At, ²¹²Pb and ²¹²Bi. Example of radioisotopes useful inimaging procedures include: ^(43K), ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁸¹Rb/^(81M)Kr, ^(87M)Sr, ⁹⁰Y, ^(99M)Tc, ¹¹¹In, ^(113M)In, ¹²³I, ¹²⁵I,¹⁸F, ⁸⁶Y, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi.

Radiolabels are conjugated to antibodies, peptides or mimetics by avariety of well-known techniques readily performed without undueexperimentation by those having ordinary skill in the art. Radiolabelsretain their radioactivity irrespective of conjugation. Conjugation maybe accomplished directly between the antibody, peptide or mimetic andthe radioisotope or linking, intermediate molecular groups may beprovided between the antibody, peptide or mimetic and the radioisotope.Crosslinkers are particularly useful to facilitate conjugation byproviding attachment sites for each moiety. Crosslinkers may includeadditional molecular groups which serve as spacers to separate themoieties from each other to prevent either from interfering with theactivity of the other. Often imaging can be imaged using fluorescein,which are activated by light. (e.g. fluorescein (green), phycoerythrin(orange), P-E-cyanine-5 (red), P-E-texas red (red), cyanine-3 (orange),cyananine-5 (red), AMCA (ultraviolet detection)

One having ordinary skill in the art may conjugate an antibody, peptideor mimetic to a chemotherapeutic drug using well-known techniques. Forexample, Magerstadt, M. Antibody Conjugates and Malignant Disease.(1991) CRC Press, Boca Raton, USA, pp. 110-152) which is incorporatedherein by reference, teaches the conjugation of various cytostatic drugsto amino acids of an antibody, peptide or to a mimetic. Such reactionsmay be applied to conjugate chemotherapeutic drugs to the antibody,peptide or mimetic. Antibodies such as peptides which have a free aminogroup may be conjugated to active agents at that group. Most of thechemotherapeutic agents currently in use in treating cancer possessfunctional groups that are amenable to chemical crosslinking directlywith proteins. For example, free amino groups are available onmethotrexate, doxorubicin, daunorubicin, cytosinarabinoside, cis-platin,vindesine, mitomycin and bleomycin while free carboxylic acid groups areavailable on methotrexate, melphalan, and chlorambucil. These functionalgroups, that is free amino and carboxylic acids, are targets for avariety of homobifunctional and heterobifunctional chemical crosslinkingagents which can crosslink these drugs directly to the single free aminogroup of a antibody of the invention.

Administration of Pharmaceutical Compositions

The dosage of the compositions of the present invention administeredwill, of course, vary depending upon known factors such as thepharmacodynamic characteristics of the particular agent, and its modeand route of administration; age, health, and weight of the recipient;nature and extent of symptoms, kind of concurrent treatment, frequencyof treatment, and the effect desired. Usually a daily dosage of activeingredient can be about 0.001 to 1 grams per kilogram of body weight, insome embodiments about 0.1 to 100 milligrams per kilogram of bodyweight. Ordinarily dosages are in the range of 0.5 to 50 milligrams perkilogram of body weight, and preferably 1 to 10 milligrams per kilogramper day. In some embodiments, the pharmaceutical compositions are givenin divided doses 1 to 6 times a day or in sustained release form iseffective to obtain desired results. In some preferred embodiments,about 5 μg to 5000 mg of antibody, peptide or mimetic may beadministered. In some preferred embodiments, 50 μg to 500 mg ofantibody, peptide or mimetic may be administered. In other preferredembodiments, 500 μg to 50 mg of antibody, peptide or mimetic may beadministered. In a preferred embodiment, 5 mg of antibody, peptide ormimetic is administered.

Compositions may be administered by an appropriate route such as, forexample, by oral, intranasal, intramuscular, intraperitoneal orsubcutaneous administration. In some embodiments, intravenousadministration is preferred. In some embodiments, the composition isadministered by intraarterial, intradermal, parenteral, or intratumoraladministration. According to some preferred embodiments, the individualhas had surgery to remove bulk tumor mass prior to administration of thecomposition.

According to some embodiments of the invention, the pharmaceuticalcompositions are administered locally at the site of the tumor. In someembodiments, the pharmaceutical compositions are administered directlyinto the tumor cells and the tissue immediately surrounding the tumor.In some embodiment, the pharmaceutical compositions are delivered intobrain tumors such as, for example, glioblastomas. In some embodiments,the pharmaceutical compositions are delivered into brain tumors as partof the surgical resection of the tumor. In some embodiment, thepharmaceutical compositions are delivered into brain tumors usingstereotaxic surgical techniques.

Subsequent to initial administration, individuals may be boosted byreadministration. In some preferred embodiments, multipleadministrations are performed.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

Because conjugated antibody, peptide or mimetic are specificallytargeted to cells with erbB, TNF, or IgSF receptors, conjugatedantibody, peptide or mimetic which comprise chemotherapeutics or toxinsare administered in doses less than those which are used when thechemotherapeutics or toxins are administered as unconjugated activeagents, preferably in doses that contain up to 100 times less activeagent. In some embodiments, conjugated antibody, peptide or mimeticwhich comprise chemotherapeutics or toxins are administered in dosesthat contain 10-100 times less active agent as an active agent than thedosage of chemotherapeutics or toxins administered as unconjugatedactive agents. To determine the appropriate dose, the amount ofantibody, peptide or mimetic is preferably measured in moles instead ofby weight. In that way, the variable weight of different antibodies,peptides or mimetics does not affect the calculation. For example,presuming a one to one ratio of antibody to active agent in conjugatedcompositions of the invention, fewer moles of conjugated antibodies maybe administered as compared to the moles of unconjugated antibodiesadministered, preferably up to 100 times less moles.

For parenteral administration, the antibody, peptide or mimetic can beformulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle.Examples of such vehicles are water, saline, Ringer's solution, dextrosesolution, and 5% human serum albumin. Liposomes and nonaqueous vehiclessuch as fixed oils may also be used. The vehicle or lyophilized powdermay contain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field which is incorporated by reference in itsentirety.

For example, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution.

The antibody, peptide or mimetic may be administered to a tissue of anindividual topically or by lavage. The antibodies, peptides or mimeticsmay be formulated as a cream, ointment, salve, douche, suppository orsolution for topical administration or irrigation. Formulations for suchroutes administration of pharmaceutical compositions are well known.

The present invention is not intended to be limited by any theory. Thepresent invention is further illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLES Example 1 The Cysteine Rich Subdomain is Responsible forDimerization of Either Homomeric or Heteromeric erbB Species

The process of homodimerization and heterodimerization in the erbBsystem has been described (Wada, et al. 1990, Cell, 61:1339-1347).Intermolecular association and trans-phosphorylation of receptormolecules result in activated heterodimeric kinase complexes which mayprovide qualitative or quantitative differences in phosphotyrosine sitesnecessary for cellular substrate binding. The basic features ofheterodimerization of p185 with the EGFr have now been found for othermembers of this family. Both erbB3 and erbB4 heterodimerize withHER2/neu indicating that this feature represents a general mechanism ofincreasing complexity and diversification of receptor function. Dimersmay nucleate the formation of tetramers or even more complex assemblies.

Dimerization studies were conducted as described in (Kumagai et al.,2001). Derivatives of p185neu were constructed, to determine whichsubdomains were required for homo- and heterodimerization. A series ofdeletion mutants of ectodomain forms of p185neu were created thatincluded a portion of the ectodomain, the transmembrane region, and afew residues of the endodomain linked to a tag (FIG. 1A). Transfectioninto Cos7 cells of all the mutant species led to comparable expression.The lysates of cells transfected with the neu species and EGFr werelysed and immunoprecipitated with antibodies specific for the His tagfound on the neu species (or, for lane 1, anti EGFr) and then blottedwith antibodies specific for the VSVG (vesicular stomatitis virusglycoprotein) tag placed on the EGF receptor (top panel) and the Myc tagon the neu receptor (middle panel). Constructs composed only ofsubdomain W associated with the EGFr holoreceptor (FIG. 1B, lane 4). Afragment of 46 amino acids of subdomain IV, (6CN lane 7) that includesthe distal 6 cysteines as its ectodomain, also associates with the EGFr.

In the schematic diagram given in FIG. 1A, numbers refer to amino acidpositions from the first Met at N-terminal of Neu or EGFr. Otherreferences are as follows: SP, signal peptide; I, subdomain I; II,subdomain II; III, subdomain III; W, subdomain IV; TM, transmembranedomain; hatched squares, TM with single-point mutation (V664G) intransmembrane domain; TK, tyrosine kinase domain; Myc, Myc epitope; His,polyhistidine tag; VSVG, VSVG tag; broken line, deleted region. In FIG.1B immunoblots (113) were performed with the antibodies as describedinfra. The membrane in the upper panel was blotted with anti-VSVG andthen reblotted with anti-Myc as indicated in the middle panel. The sametotal cell lysates were subjected to a nitrocellulose membrane (bottompanel) in order to determine the exogenous expression of EGFr (supra andinfra).

Other studies compared the ability of EGF to stimulate MAP kinaseactivation by the EGFr when these variant p185neu species were alsoexpressed. It was found that the pTex6CN form was able to associate withthe EGFr and inhibit the kinase activation of the EGFr. (Kumagai et al.,2001). The terminal 46 amino acids region is composed of cystine knots.These studies emphasize the critical nature of the cystine knotcontaining regions of erbB as essential for the assembly of erbBreceptor complexes.

These results suggested that small forms of cystine knot mimetics mightbe used as antagonists of receptor oligomerization. These results alsoprovide the framework for developing the new class of MAb reactive withthis region of p185 as a means to disable receptor associations as a newapproach to preempt or disrupt transforming complexes.

Example 2 erbB Subdomain IV Cystine Knot Mimetics

A series of peptides was designed to mimic potential dimerization sitesin the C-terminal part of the S2 domain of different erbB receptorsusing molecular models of the dimeric complexes. The peptides weredesigned based on the molecular model of the subdomain W of erbBreceptors constructed by comparative modeling with the second subdomainof the type-1 insulin-like growth factor receptor (IGF-1R), as well asstructures of the TNF receptor and laminin that have similar disulfidebond connectivities (Garrett, et al. 1998, Nature, 394:395-9; Naismith,et al. 1995, J. Biol. Chem., 270:13303-13307; Naismith and Sprang 1998,Trends Biochem Sci, 23:74-9).

The following peptides were constructed:

B1-S22-ALG (derived from erbB1) YCLVWKYADAGCY; (SEQ ID NO: 32)B2-S22-APE (derived from erbB2) YCPIWKIFPDEECY; (SEQ ID NO: 31)B2-S22-AFA (derived from erbB2) YCFPDEEGACY; (SEQ ID NO: 35) B2-S23-BPT(derived from erbB2): PCPINCTHSCVDLDDKGCPAEQRASPLTSI; (SEQ ID NO: 38)B3-S22-APQ (derived from erbB3) YCPIYKYPDVQCY; (SEQ ID NO: 33) andB4-S22-AFD (derived from erbB4) YCFIFKYADPDCY. (SEQ ID NO: 34)

Peptides B1-S22-ALG, B2-S22-APE, B2-S22-AFA, B3-S22-APQ and B4-S22-AFDmimic the S22 repeat. B2-S23-BPT was derived from the membrane-proximalS23 repeat. A CD4 receptor-derived cyclic peptide, CD4-G (FCYIGEVEDQCY;SEQ ID NO: 43), was designed as a negative control.

FIG. 2 shows the molecular models of the fourth subdomain of HER2 andmimetic peptides derived from the C-terminal part of this subdomain.B2-S22-AFA is a cyclic peptide that mimics part of the S22 loop.B2-S23-BPT is a bicyclic peptide constrained by two disulfide bonds andrepresents a whole S23 repeat followed by the juxtamembrane amino acidresidues. Molecular modeling indicates close conformational similaritybetween the constrained mimetic peptides and corresponding loops of thereceptor. Based on the existing structural and experimental data, apossible arrangement of the receptor has been proposed, and the S22domain is suggested as a major inter-receptor interaction site withinthe complex.

Example 3 Kinetic Binding Analysis of the S22 Peptide

The designed erbB peptides were tested for binding to erbB receptorsusing surface plasmon resonance (Biacore) technology (described supra)using soluble erbB-1 and erbB-3. The response was plotted in real timein the form of sensorgram curves. Dissociation constants (K_(d)) weredetermined as described (Park, et al. 2000, Nat. Biotechnol.18:194-198).

A sensorgram for binding of B2-S22-APE peptide to erbB receptorsimmobilized on the sensor chip was performed. (FIG. 3A.) Kineticconstants were estimated by global fitting analysis of the titrationcurves to the 1:1 Langmurian interaction model, which gave a k_(on) of3.24×10³ M⁻¹s⁻¹, and a k_(off) of 6.85×10⁻⁴ s⁻¹. The k_(off)/k_(on)ratio gave a value of 0.21 μM for the dissociation constant (K_(d)).Good fitting of experimental data to the calculated curves has beenobserved, suggesting a simple pseudo-first order interaction between thepeptide and the receptor.

K_(d) values for other erbB peptides, analyzed in a similar fashion asB2-S22-APE, are presented in Table 1. All erbB peptides showed bindingto different erbB receptors. However, no binding could be detected tothe immobilized TNF receptor used as a control, indicating selectivityof erbB peptides to the erbB receptor family. A control CD4-G peptidedid not bind to any of the studied receptors. Some binding specificitycould also be observed for different peptides within the erbB family.Thus, erbB2-derived peptide, B2-S22-APE could bind to the erbB1 receptorbetter than to erbB2 and erbB3. ErbB1-derived B1-S22-ALG showedpreferential binding to erbB3. ErbB2-derived B2-S22-AFA peptide did nothave a high specificity within erbB receptors, but showed the bestoverall binding affinity to all three receptors.

TABLE 1 ErbB receptor-derived peptides. K_(D) (μM) ErbB1 ErbB2 ErbB3TNFR B2-S23-BPT 2.06 4.23 0.832 >10³ B2-S22-APE 0.210 1.53 1.40 >10³B1-S22-ALG 0.449 0.371 0.288 >10³ B3-S22-APQ 0.472 0.549 1.07 >10³B4-S22-AFD 0.583 0.639 0.394 >10³ B2-S22-AFA 0.407 0.302 0.429 >10³CD4-G >10³ >10³ >10³ >10³ Binding to the immobilized extracellulardomains of the ErbB receptors studied by surface plasmon resonance. *C-terminus is blocked with NH₂

To demonstrate that erbB peptides bind to Sbd IV of erbB receptors, theinteraction of the B2-S22-APE peptide with recombinantly expressederbB2-SbdIV immobilized on the surface chip was determined (FIG. 3B.).The observed binding to erbB2-SbdIV had very similar kinetic constants(k_(on), 1.62×10³ M⁻¹s⁻¹; k_(off), 3.07×10⁻³s⁻¹) and affinity (K_(D),1.89 μM) as binding to the whole ectodomain of erbB2 (FIG. 3A; Table 1).Binding of other erbB peptides to erbB2-Sbd W was undistinguishable fromtheir binding to the full-size erbB2 ectodomain (data not shown),demonstrating that subdomain IV is the binding site for all erbBpeptides. Since erbB peptides are derived from subdomain IV of differenterbB receptors, this fact indicates direct involvement of subdomain IVin receptor-receptor interactions between the members of erbB receptorfamily.

Example 4 Inhibition of Receptor Self-Associations

Surface Plasmon Resonance Studies.

The effect of cysteine-knot mimetics on receptor self-associations, wasexamined using a Biacore assay in which the ectodomains of erbBreceptors were immobilized on the surface chip and the ectodomain oferbB3 was injected at 300 nM concentrations either alone or in thepresence of 5 μM hereglunin HRGβ1. A very limited degree ofreceptor-receptor interactions in the absence of heregulin was observed.(FIG. 4; 1A, 2A, 3A.) However, when erbB3 was preincubated with about17-fold molar excess of HRGβ1, a strong binding to all three erbBreceptors was observed (FIG. 4; 1C, 2C, 3C), indicating ligand-inducedhomo- and heteromerization of the erbB receptor ectodomains. No increasein binding upon preincubation with heregulin was observed for the TNFreceptor used as a control (data not shown). Kinetic analysis of thedose response curves (data not shown) revealed that the strongestbinding was observed for erbB3-erbB1 heteromerization (K_(D), 2.3 nM),followed by erbB3-erbB3 homomerization (K_(D), 7.2 nM) and erbB3-erbB2heteromerization (K_(D), 17.9 nM).

In a parallel experiment, it was determined whether similarligand-induced receptor self-associations could be observed for theerbB1 receptor injected in the presence of EGF or TGFα. However, nointeraction of the injected erbB1 ectodomain (150 nM) with theimmobilized receptors could be detected either in the absence or in thepresence of the erbB1 ligands (5 μM), suggesting that at this erbB1receptor concentration no significant dimerization is taking place.

The effect of the erbB peptides on heregulin-induced receptorself-associations was studied by preinjecting them at 10 μM followed byinjection of erbB3-HRGβ1 (FIG. 4; 1B, 2B, 3B). The B2-S22-AFA peptideeffectively inhibited binding of erbB3 to all three immobilizedreceptors. The highest degree of inhibition was observed for theerbB3-erbB2 heteromerization (82.5%) followed by erbB3-erbB1heteromerization (78.4%) and erbB3-erbB3 homomerization (61.7%). As acontrol, either the running buffer or a control peptide (CD4-G) werepreinjected instead of the ErbB peptides and showed no effect onreceptor self-associations (data not shown).

Values for inhibition of dimerization of different erbB peptides aregiven in Table 2. As expected from the data for the receptor-bindingaffinities, B2-S22-APE, which showed the best binding to erbB1 (Table1), was more effective in disabling interaction of erbB3 with erbB1 thanwith other receptors (Table 2). Similarly, B1-S22-ALG with the highesterbB3-binding affinity (Table 1) was the strongest inhibitor of erbB3homodimerization (Table 2). B2-S22-AFA with the strongest overallaffinity to the three studied erbB receptors (Table 1), is an effectiveinhibitor of erbB3-erbB2 and erbB3-erbB1 interactions (Table 2).B2-S23-BPT, derived from the S23 repeat of erbB2, had the lowest butstill significant inhibitory effect, especially on the homomerization oferbB3 (Table 2) to which it binds with the highest affinity compared toother receptors (Table 1).

TABLE 2 Inhibition of ligand-induced ErbB receptor dimerization by ErbBreceptor-derived peptides. Percent Inhibition^(a) ErbB1 ErbB2 ErbB3B2-S23-BPT 41.5 32.3 55.4 B2-S22-APE 80.3 51.1 42.7 B1-S22-ALG 65.2 71.475.1 B3-S22-APQ 69.0 58.1 32.4 B4-S22-AFD 60.3 54.2 72.8 B2-S22-AFA 78.482.5 61.7 CD4-G 2.4 −1.2 3.1 ^(a)Inhibitory effect of ErbB peptides (10μm) on the binding of ErbB3 (300 nm) to the immobilized ErbB receptorsin the presence of HRGβ1 (5 μM).

Inhibition of Ligand-Induced Dimerization of Native erbB Receptors in32D Cell Lines.

Effect of the ErbB peptides on biochemically defined dimerization ofnative full-length ErbB receptors was studied using the 32D cell linestransfected with different erbB receptors. 32D-E1 cells were transfectedwith erbB1; 32D-E2/E3 with erbB2 and erbB3; and 32D-E2/E4 with erbB2 anderbB4. FIGS. 5A and 5B shows the inhibitory effect of 10 μg/mlB3-S22-APQ and B2-S22-AFA on the heregulin-induced erbB2-erbB4 receptorheteromerization in the 32D-E2/E4 cells. While B3-S22-APQ showed asignificant inhibitory effect, B2-S22-AFA completely suppresseddimerization at this concentration. (FIG. 5A.) In contrast, noinhibitory effect was observed for the CD4-G peptide used as a control.The observed inhibition of receptor dimerization by B2-S22-AFA wasdose-dependent (FIG. 5B) with an apparent IC₅₀ concentration of 0.8 μM.Similar inhibitory effects of B3-S22-APQ and B2-S22-AFA on receptordimerization were observed in the 32D-E2/E3 cells (data not shown).

Example 5 Biological Activity of the erbB Peptides

MTT Assay

Biological activity of erbB peptides was evaluated by their ability toinhibit cell proliferation using standard3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) (MTT)assays, as described supra (Hansen et al., J. Immunol. Methods 1989,119, 203-210). HER2-expressing transformed tumor cells (T6-17) were usedfor this purpose. (Park et al., Nat. Biotechnol. 2000, 18, 194-198). InMTT assays, the peptides inhibited the growth of T6-17 cells, anerbB2-overexpressing transformed cell line, dose-dependently atconcentrations ranging from 0.01 to 10 μg/ml. (FIG. 6.) Biologicalactivity of erbB peptides at the optimal concentration of 1 μg/ml wasassessed. Each value represents an average of at least four samples. Allpeptides show inhibitory effects on cell growth. (FIG. 6.) B2-S22-AFA,which has the highest erbB2 receptor-binding affinity (Table 1), wasalso the most active peptide in the MTT assay. (FIG. 6.)

To determine if the observed biological activities of erbB2 peptidesagainst T6-17 cells correlated with their receptor-binding properties ortheir inhibitory activity on receptor-receptor interactions, the bindingdata shown in Table 1 and Table 2 were plotted against the MTT assaydata for all studied peptides. (FIGS. 7A and 7B.) There was a strongcorrelation (r=0.92) between the biological activity and bindingaffinity to erbB2. Correlation with binding affinities to other erbBreceptors (erbB1 and erbB3) was insignificant (r=0.21 and 0.44,respectively). Similarly, a strong correlation (r=0.91) has beenobserved between the cell-suppressing activity and inhibitory activityagainst erbB3-erbB2 interactions, but not against erbB3-erbB1 (r=0.29)or erbB3-erbB3 (r=0.17) interactions (FIG. 7B).

Effect on the Viability of 32D Cell Lines.

Inhibitory effects of the erbB peptides on cells over-expressing erbBreceptors was tested using the 32D cell lines. Transfection of 32D cellswith erbB receptors created transfectants capable of growing in a mediumcontaining either EGF (32D-E1), HRGβ1 (32D-E2/E3 and 32D-E2/E4) or IL3(supplement WEHI medium, all three cell lines). (Wang et al., Proc.Natl. Acad. Sci. USA 1998, 95, 6809-6814.) FIG. 8A shows the effect ofdifferent erbB peptides on the viability of the 32D transfectants grownin the erbB ligand medium. The strongest inhibitory effect was observedfor the 32D-E1 cells (grown in the EGF medium). Effect on the 32D-E2/E3and 32D-E2/E4 cells (grown in the HRGβ1 medium) was less pronounced butalso significant (FIG. 8A). However, when same cell lines were grown inthe IL3 supplement WEHI medium, no significant effect on cell viabilitycould be detected for any of the studied peptides (FIG. 8B), confirmingthat the observed inhibition of cell survival by the ErbB peptides ismediated by their specific effect on the ErbB receptor signaling but noton IL-3-related signaling.

ErbB peptides described in this study represent constrained mimics ofthe loops or repeats present in the subdomain W of the erbB receptorsand based on our molecular modeling studies, show a high degree ofstructural similarity with the template receptor regions. Theinteractions observed for the erbB peptides are also displayed by thecorresponding sites of the native erbB receptors and play a certain rolein their metabolic activity. Information obtained for the B2-S23-BTEpeptide is especially valuable. Unlike other designed erbB peptides, itdoes not mimic a single loop, but rather represents a C-terminalmembrane-proximal portion of the erbB2 ectodomain. Thus, since theC-terminal portion of erbB2 (B2-S23-BPT) can bind to all three studiederbB receptors (Table 1), this property is also likely to be expectedfrom the full-length native erbB2 receptor. The fact that erbB peptidesare not specific to any particular erbB receptor but are highly specificto the erbB family (Table 1), suggests that the C-terminal part ofsubdomain W is a receptor-receptor interaction site shared by all erbBreceptors. Since all erbB peptides could bind to their respectiveparental receptor and to other erbB receptors (Table 1), these sites canparticipate in both homo- and heteroreceptor self-associations.

The observed inter-receptor interactions involving the C-terminalportion of subdomain W are important for receptor function. Theinhibition by the erbB peptides resulted in a dramatic suppression ofthe cell growth. Significant correlation was found between peptidesbinding to erbB2, their inhibition of erbB3-erbB2 binding, and theirinhibitory activity against erbB2-overexpressing T6-17 cells in the MTTassay.

The observed biological activities of erbB peptides were mediated bytheir binding to the erbB2 receptor and by blocking receptor-receptorinteractions that involve erbB2. erbB peptide-induced inhibition of cellgrowth in 32D cells transfected with erbB receptors has also been shownto be specifically mediated by the erbB receptor pathway. Indeed, stronginhibition of cell growth has been observed only when the cells weregrown in the erbB ligand medium. In contrast, no inhibition occurred viaan erbB receptor-independent pathway when the cells were grown in theIL3 supplement (WEHI) medium.

In summary, erbB receptor signaling can be inhibited by rationallydesigned interface peptide mimetics derived from the subdomain IV oferbB receptor ectodomains. The mimetics specifically bind to thereceptors of the erbB family and block inter-receptor interactions whichleads to the growth inhibition of HER2-overexpressing cells in vitro.Since all four erbB receptors represent a therapeutic target, peptidemimetics that selectively bind to this receptor family and disable theiractivity could have an advantage over drugs that are specific to asingle member of the erbB family. The study also demonstrates theimportance of the C-terminal part of subdomain IV for receptor-receptorinteractions involved in signaling by erbB family members.

Example 6 Solubility and Stability of S22-AFA Analogs

Enhancement of solubility of S22-AFA analogs can be accomplished bymodifying certain residues that should not affect activity. Thefollowing changes (boldface) are made to increase the solubility:

(1) YCF(Y)PDEEGACY-OH (SEQ ID NO: 3; SEQ ID NO: 12) (2) YCFPDEEGACYK-NH₂ (SEQ ID NO: 25) (3) YCFPDEEGACYGGS (SEQ ID NO: 26) (4)GGSYCFPDEEGACY-NH₂ (SEQ ID NO: 6)

Example 7 Creation of Mab which Bind to Interaction Surfaces

MAb specific to dimerization domains are made from: (1) Recombinantlypurified p185 subdomain IV fragment; (2) Improved S22-AFA analogs; and(3) other immunogens. Using both the subdomain IV fragment, andcysteine-knot peptides will yield high quality cross-reactive MAb. Thesespecies are used to immunize Balb/c mice to create a specific dimersurface inhibitory monoclonal species. S22-AFA is coupled to a carrierspecies as described previously (Jacob, et al, 1985; Williams, et al,1989, J. Immunol., 142: 4392-4400; Christodoulides, et al, 1993, J.Genetic Microbiology, 139:1729-1738). The subdomain IV fragment is usedas is. Mab production employs a scheme described previously (Drebin, etal. 1986, Symp Fundam Cancer Res, 38:277-289; Drebin, et al. 1986, ProcNatl Acad Sci USA 83:9129-9133). Briefly, BALB/c (H-2^(d)) mice will beimmunized with 100 μg of the most efficacious S22-AFA species or solublesubdomain W (in equal volumes of complete Freund's adjuvant)subcutaneously and then boosted 3 times intraperitoneally with 50μg/injection. Three days after the final boost, fusions are performedusing spleen cells and the fusion partner Sp2/0-Ag14. Screening offusions will employ an ELISA with S22-AFA deposited in the wells. Otherscreening assays to be used include FACS analysis of T6-17 cellsexpressing HER2/neu.

MAb are generated and selected for their ability to bind S22-AFA peptideforms and to bind to Her2/neu on cells and are evaluated functionally invitro in anchorage independent and dependent type studies.

MAb are evaluated for in vivo effects on tumors using EGFr, Her2/neu,and EGFr and Her2/neu transformed cells (see above). 100 μg MAb isadministered by intraperitoneal injection three times a week from theday of tumor xenograft. Injection of irrelevant anti-CD4 MAb or PBSserves as a control Inhibiting the formation of oligomeric receptorforms will affect phenotype. The effect of co-treatment withdoxorubicin/adriamycin which has been shown an increased effect on cellstreated with antibodies (Park et al., Nat. Biotechnol. 2000, 18,194-198) is also examined. Tumor growth is monitored by volumemeasurement.

Example 8 Development of Cystein Rich Domain (CRD) Reactive MonoclonalAntibodies

Cell lines transfected with erbB constructs are used as immunogens tocreate cysteine rich domain reactive MAb that are compared with themimetics in terms of biological activity. NR6 cells transfected withpTex3-4, pTex4 and pTec6CN (See FIG. 1A and Kumagai et al, 2001) areused as immunogens. The fusion scheme is as described previously(Drebin, et al. 1986, Symp Fundam Cancer Res 38:277-289; Drebin, et al.1986, Proc Natl Acad Sci USA 83:9129-9133). Briefly, BALB/c (H-2^(d))mice are immunized with the cell line subcutaneously and then boosted 3times intraperitoneally with 10⁷ cells. Three days after the finalboost, fusions are performed using spleen cells and the fusion partnerSp2/0-Ag14. The SP2/0-Ag14 fusion partner secretes no free light chain.Hybridoma cell lines will be screened by FACS analysis against pNeu,pTex3, pTex3-4 (subdomains III and IV), pTex4 (subdomain IV only) andpTex6CN. Controls include NR6 cells, cells which express EGFr alone andpNex1, 2, and 3 (subdomains I, II, and III) and a cell line thatexpresses only subdomain I, pTex1 cells. Cell lines provide anunambiguous screening array for this class of MAb. Colonies producingantibodies of the desired specificity are subcloned three times bylimiting dilution. Subtypes for MAb are identified using the MouseMonoclonal Antibody Subtyping Kit (Gibco BRL).

Experimental Procedures.

Peptide Synthesis and Cyclization

Linear peptides (95% purity) were ordered from the Protein ChemistryLaboratory, University of Pennsylvania. Peptide purity and identity wasconfirmed by reverse phase high performance liquid chromatography (RPHPLC) and MALDI mass-spectrometry, using a time-of-flight massspectrometer (MicroMass TofSpec; Micromass Inc., Beverly, Mass.). Thepeptides were cyclized by air oxidation in distilled water adjusted topH 8.0 with (NH₄)₂CO₃ at 0.1 mg/ml and 4° C. Progress of the oxidationwas controlled by measuring amounts of free thiols with5,5′-dithio-bis(2-nitrobenzoic acid (DTNB). Briefly, 0.4 ml of a peptide(0.1 mg/ml) and 5 μl of DTNB (20 mM) were added to 0.2 ml of 0.1M sodiumphosphate buffer, pH 8.0. Absorbance at 412 nm was measured and comparedwith the linear unoxidized peptides. The cyclized peptides werelyophilized and their purity analyzed by RP HPLC using a C18semi-preparative column (Waters, Milford, Mass.). Typically, purity ofhigher than 95% was obtained for the cyclized peptides. Aliquotes of 1mM stock solutions have been prepared for each peptide and kept at −20°C. to be thawed prior to the binding or bioassay studies. Peptideconcentrations were confirmed by UV spectrofotometry using extinctioncoefficients at 280 nm calculated for each peptide as described in Gillet al., (Anal Biochem 1989, 182, 319-326).

Expression of the GST Fusion Protein of Subdomain IV of ErbB2.

The DNA fragment encoding the subdomain W of erbB2 (erbB2-SbdIV) wasgenerated by polymerase chain reaction. The upstream primer was5′-CGCCCGGATCCTGGCCTGCCACCAGCTGTGC-3′ [SEQ ID NO: 44] and the downstreamprimer was 5′-CGCCCGCGGCCGCCGCAGAGATGATGGAGTCAG-3′. [SEQ ID NO: 45]These two primers were designed to include BamHI and Not I restrictionsites, respectively, for inframe insertion into the BamHI/Not Ilinearized pGEX-5X-3 vector. Recombinant vector was used to infectEscherichia coli BL-21 (DE3). 100 ml of the 2XYT medium were inoculatedwith 10 ml of the overnight culture and grown at 37° C. until the OD₆₀₀of 0.4-0.5 was reached. IPTG was added to the medium at the finalconcentration of 0.1 mM and grown for 2 hrs. The cells were spun down bycentrifugation at 4,000 g for 10 min and resuspended with 5 ml of coldPBS (with DTT, PMSF and aprotinin at the final concentration of 5 mM, 1mM and 1 μg/ml respectively). After sonication on ice, Triton X-100 wasadded to the final concentration of 1% and the solution was rocked at 4°C. for 1 hr and centrifuged at 10,000 g for 10 min. 100 μl ofglutathione sepharose bead was then added to the supernatant. It wasrocked at 4° C. for 2-4 hrs, centrifuged and washed three times withPBS. 100 μl elution buffer was added followed by rotation for 10 min atroom temperature, centrifugation at 300 g for 1 min and collection ofthe supernatant. Elutions were repeated three times and combined.

Interaction Studies

Binding experiments were performed with the surface plasmon resonancebased biosensor instrument Biacore 3000 (Biacore AB, Uppsala, Sweden),at 25° C. Recombinant purified erbB receptors were provided by Dr. CheLaw, Xcyte Therapeutics, Seattle, Wash. (erbB2) and Dr. Mark A. Lemmon,Department of Biochemistry and Biophysics, University of PennsylvaniaSchool of Medicine (erbB1 and erbB3). Immobilization of the erbBreceptors in the sensor surface was performed following the standardamine coupling procedure according to manufacturer's instructions.Briefly, 35 μl of a solution containing 0.2 MN-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC) and 0.05 MN-hydroxysuccinimide (NHS), were injected at a flow rate of 5 μl/min toactivate carboxyl groups on the sensor chip surface. Receptors (40 ng/mlin 10 mM NaOAc buffer, pH 5.0) were flowed over the chip surface at aflow rate of 20 μl/min until the desired level bound protein wasreached. Unreacted protein was washed out and unreacted activated groupswere blocked by the injection of 35 μl of 1 M ethanolamine at 5 μl/min.The final immobilization response of each receptor was 3,500 RU. Areference surface was generated simultaneously under the same conditionsbut without receptor injection and used as a blank to correct forinstrument and buffer artifacts. Peptides were injected at variableconcentrations at 20 μl/min flow rate and binding to the receptorsimmobilized on the chip was monitored in real time. Each sensorgramconsists of an association phase (first 240 s), reflecting binding ofthe injected peptide to the receptor, followed by a dissociation phase(300 s), during which the running buffer is passed over the chip and thebound peptide is being washed off the receptor surface.

MTT Assay

The MTT assay has been used for measuring cell growth as previouslydescribed in Hansen et al (J. Immunol. Methods 1989, 119, 203-210).Briefly, T6-17 cells were seeded in 96-well plates overnight in DMEMcontaining 10% FBS (1000 per well). T6-17 is derived from N1H3T3 byoverexpressing the human erbB2 receptor. Cells were cultured in 100 μlof fresh medium containing 1 μg/ml of erbB peptides for 48 hours. Thisincubation time was optimal for measuring inhibitory effects ofdifferent analogs. No improvements in the inhibitory activity could beachieved by increasing the incubation period. 25 μl of MTT solution (5mg/ml in PBS) were added to each well, and after 2 hours of incubationat 37° C., 100 μl of the extraction buffer (20% w/v of SDS, 50%N,N-dimethyl formamide, pH 4.7) were added. After an overnightincubation at 37° C., the optical density at 600 nm was measured usingan ELISA reader.

Cell Viability and Crosslinking Analysis on 32D Cell Lines.

32D cell transfectants with ErbB receptors (gift from Dr. Jacalyn H.Pierce, National Cancer Institute) were grown in RPMI 1640, 10% FBS and5% WEHI medium (GenoQuest, Interlukin-3 supplement) and respectiveantibiotics, i.e. 32D-E1 (gptr), 32D-E2/E3 (neor/gptr) and 32D-E2/E4(neor/gptr) (29). The WEHI medium was withdrawn and cells werepre-incubated with ErbB peptides for 2 hours at 37° C. before adding 10μg/ml EGF (for 32D-E1, Collaborative Biomedical Products) or 10 μg/mlHRG□1 (for 32D-E2/E3 or 32D E2/E4, R&D Systems) and further incubated at37° C. for 24-48 hours. The cell viability was detected with propidiumiodide staining followed by flow cytometry quantification. For thecross-linking analysis, approximately 2×106 cells were suspended in RPMI1640 medium containing 0.1% BSA and 10 mM HEPES, and pre-incubated withErbB peptides for 2 hours at 37° C. before adding 10 μg/ml EGF or 10μg/ml Heregulin-β1 EGF domain and further incubated at 37° C. for 10-15minutes. Cells were rinsed with PBS and incubated in 2 mM BS³/PBS at 4°C. for 45 minutes. Cell lysate was immunoprecipitated with anti-ErbB2 oranti-ErbB3 antibody (Santa Cruz), and immunobloted with anti-pTyr (PY20,Santa Cruz).

Model Building.

Homology modeling of erbB2-SbdIV and erbB1 ectodomain was performed withQuanta/Protein design (Molecular Simulations Inc.) on the basis oftemplate crystal structures of laminin g1 III3-5 (1KLO) for erbB2-SbdIVand insulin-like growth factor-1 receptor (1IGR) for erbB1. Thesequences were aligned manually using the Sequence Viewer by matchingpositions of conservative cysteine residues and inserting gaps to adjustthe lengths of the inter-cysteine spacings. Frameworks for the molecularmodels were generated by using coordinates from the template structuresfor manually selected matching residues of the modeled proteins. Missingcoordinates for peptide segments that did not have counterpart in thetemplate structures were calculated by either “Regularizing Region” and“Model Side Chains” tools (for short loops) or by modeling loopconformation using a “Congen” (Li et al., 1997, Protein Sci., 6,956-970; Tejero, R., et al., 1996, Protein Sci., 5, 578-592) program(for longer loops). The final monomeric structures were then obtained byrunning the CHARMm energy minimization in the RTF mode. To construct adimeric erbB1-EGF model, the following assumptions were made based onthe existing experimental evidence: erbB1-EGF complex has a 2:2stoichiometry (Lemmon M. A., et al., 1997, EMBO J., 16, 281-294); theC-terminal part of subdomain IV is a dimeric interaction site (based onour results described below); the N-terminus of bound EGF is close(within about 15 Å) to Tyr101 (subdomain I) of erbB1 (Woltjer, R. L., etal., 1992, Proc. Natl. Acad. Sci. USA, 89, 7801-7805; the C-terminalArg45 of bound EGF is close (within about 15 Å) to Lys465 (subdomainIII) of erbB1 (Summerfield, A. E., et al., 1996, J. Biol. Chem., 271,19656-19659; the N-terminus of EGF bound to erbB1 is about 67 Å (from 52to 82 Å) away from the membrane surface (Carraway, K. L., 1990,Biochem., 29, 8741-8747; maximal dimensions of erbB1 are about 110 Å forthe monomer and 120 Å for the dimer (Tejero, R., et al., 1996, ProteinSci., 5, 578-592); EGF binds to the second face of subdomain III(Jorissen, R. N., 2000, Protein Sci., 9, 310-324. Orientations ofcomplex-forming two erbB1 and two EGF (PDB code, 3EGF) molecules wereadjusted manually to satisfy the criteria listed above based on twodifferent models of the complex arrangement. The final dimeric modelswere minimized using the CHARMm energy minimization tool. These dimericcomplex models have a low resolution nature and may have a degree oferror in the positioning of the constituent molecules with respect toeach other.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

The entire disclosure of each patent, patent application, or otherpublication cited herein is hereby incorporated by reference.

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Each reference cited herein is hereby incorporated by reference in itsentirety.

1. An isolated peptide having the formula:R₁-R₂-R₃-R₄-R₅-R₆-R₇ wherein: R₁ is F, Y, F-G, or Y-G; R₂ is cysteine orpenicillamine; R₃ is a bond; R₄ is F-P-D-E-E-G-A (SEQ ID NO:1) orF-Y-P-D-E-E-G-A (SEQ ID NO:2); R₅ is a bond; R₆ is cysteine orpenicillamine; and R₇ is F or Y; and wherein: R₁, R₂, R₃, R₄, R₅, R₆ andR₇, taken together, comprise 30 amino acids or less.
 2. The peptide ofclaim 1, wherein R₄ is F-P-D-E-E-G-A (SEQ ID NO:1).
 3. The peptide ofclaim 1, wherein R₄ is F-Y-P-D-E-E-G-A (SEQ ID NO:2).
 4. The peptide ofclaim 1, wherein: R₁ is Y, F-G, or Y-G; and R₄ is F-Y-P-D-E-E-G-A (SEQID NO:2).
 5. The peptide of claim 1, wherein: R₁ is Y, F-G, or Y-G; andR₄ is F-P-D-E-E-G-A (SEQ ID NO:1).
 6. The peptide of claim 1, whereinsaid peptide comprises an amino acid sequence of Y-C-F-P-D-E-E-G-A-C-Y(SEQ ID NO: 35).
 7. The peptide of claim 1, wherein R₂ and R₆ areconnected by a bond to cyclize said peptide.
 8. An isolated peptidecomprising an amino acid sequence ofC-K-V-E-L-M-Y-P-P-P-Y-F-V-G-M-G-N-G-T-Q-I-Y-V-I-D-P-E-P-C (SEQ IDNO:28).
 9. An isolated peptide comprising an amino acid sequence ofC-K-I-E-F-M-Y-P-P-P-Y-L-D-N-E-R-S-N-G-T-I-I-H-I-K-E-K-H-L-C (SEQ ID NO:29).
 10. An isolated peptide comprising an amino acid sequence ofC-S-L-S-I-F-D-P-P-P-F-Q-E-R-N-L-S-G-G-Y-L-H-I-Y-E-S-Q-L-C (SEQ ID NO:30).
 11. An isolated peptide having an amino acid sequence ofY-C-P-I-W-K-F-P-D-E-E-C-Y (SEQ ID NO: 31); Y-C-L-V-W-K-Y-A-D-A-G-C-Y(SEQ ID NO: 32); Y-C-P-I-Y-K-Y-P-D-V-Q-C-Y (SEQ ID NO: 33); orY-C-F-I-F-K-Y-A-D-P-D-C-Y (SEQ ID NO: 34).
 12. An isolated peptidecomprising an amino acid sequence ofP-C-P-I-N-C-T-H-S-C-V-D-L-D-D-K-G-C-P-A-E-Q-R-A-S-P-L-T-S-I (SEQ ID NO:38).